Specific expression of Anaplasma marginale major surface protein 2 salivary gland variants occurs in the midgut and is an early event during tick transmission

Abstract

Infectivity of Anaplasma spp. develops when infected ticks feed on a mammalian host (transmission feed). Specific Anaplasma marginale major surface protein 2 (MSP2) variants are selected for within the tick and are expressed within the salivary glands. The aims of this study were to determine when and where MSP2 variant selection occurs in the tick, how MSP2 expression is regulated in salivary glands of transmission-feeding ticks, and whether the number of A. marginale organisms per salivary gland is significantly increased during transmission feeding. The South Idaho strain of A. marginale was used, as MSP2 expression is restricted to two variants, SGV1 and SGV2, in Dermacentor andersoni. Using Western blot, real-time PCR, and DNA sequencing analyses it was shown that restriction and expression of MSP2 occurs early in the midgut within the first 48 h of the blood meal, when ticks acquire infection. A. marginale is present in the tick salivary glands before transmission feeding is initiated, but the msp2 mRNA and MSP2 protein levels per A. marginale organism increase only minimally and transiently in salivary glands of transmission-feeding ticks compared to that of unfed ticks. A. marginale numbers per tick increase gradually in salivary glands of both transmission-fed and unfed ticks. It is concluded that MSP2 variant selection is an early event in the tick and that MSP2 variants SGV1 and SGV2 are expressed both in the midgut and salivary glands. While MSP2 may be required for infectivity, there is no strict temporal correlation between MSP2 expression and the development of infectivity.

FIG. 1.

MSP2 variants in the blood and tick organs during acquisition and transmission feeding of D. andersoni. (A) Representation of the msp2 operon indicating the expression site structure and primer pair positions. HVR indicates the location of the hypervariable region of msp2 (11); P indicates the location of the operon promoter. Numbering of nucleotides is based on ORF2 and the pCKR11.2 msp2 sequences (24). (B) PCR analysis for full-length msp2 in ticks. Ticks were acquisition fed (A) on calf 873, held at 26°C (H), and transmission fed (T) on calf 882. Midguts (MG) were collected before ticks were attached (0d), on days 1 through 6 during the acquisition feeding (1d through 6d), days 3 and 7 (3d and 7d) of the holding period, and day 3 (3d) of the transmission feeding. Salivary glands (SG) were obtained on day 3 of the transmission feeding. Blood (blood) was collected from calf 873 on day three during the acquisition feeding. The reactions were carried out with primer pair MSP2:184-201 and MSP2:891-874. A reaction mixture without template served as negative control (H₂O). Amplicons were identified by agarose gel electrophoresis and ethidium bromide staining. The position of the 700-bp fragment is indicated in the left margin. (C) Amino acid sequence alignment of MSP2 expressed in the blood of calf 873 during acquisition feeding of the ticks and in midgut samples from days 1 (1d A), 2 (2d A), and 6 (6d A) of the acquisition feeding. Only one sequence of each type obtained by cloning at each time point is shown. Sequences are compared to the published sequences of the tick-specific MSP2 variants SGV1 and SGV2 (27) and to the sequence of pseudogene A3 (Psd A3) (3). The first depicted amino acid corresponds to amino acid 201 (aa 201) of full-length MSP2 (24). The asterisk designates corresponding clones obtained from the tick midgut and blood sample.

FIG. 2.

Expression of MSP2 variants SGV1 and SGV2 from the operon site. (A) PCR analysis for operon-linked msp2 performed on midguts (MG) and salivary glands (SG) obtained at day 4 of the acquisition feeding (A) on calf 873 and day 3 of the transmission feeding (T) on calf 882. Blood was collected from calf 873 on day 3 during the acquisition feeding (blood). Reactions were carried out using the primer pair ORF2:261-282 and MSP2:891-874. A reaction mixture without template served as negative control (H₂O). Amplicons were electrophoresed using agarose gels and were stained with ethidium bromide. The position of the 1,100-bp fragment is indicated in the right margin. Amplicons from tick salivary glands and midguts were cloned and sequenced and confirmed to be SGV1 and SGV2 (data not shown). (B) Western blot analysis of midgut samples obtained from ticks acquisition fed for 8 days on calf 839 (A) and held for 3 or 7 days at 26°C (H). MSP2 was detected with a polyclonal serum, R883, against purified native MSP2 (24). MSP5 was used to control for equal loading of organism numbers, and MSP5 protein was detected by using monoclonal antibody ANAF16C1 (16). The positions of SGV1, SGV2, and MSP5 are indicated in the left margin, and molecular size markers are indicated in the right margin.

FIG. 3.

Quantitative real-time PCR for msp2 DNA. Standard curve generated with serial dilutions of a pBS vector containing full-length msp2 (24). The log calculated copy number of plasmid DNA (Log CO; x axis) is plotted against the corresponding threshold cycle (Ct; y axis). The slope, intercept, and correlation are indicated. Efficiency was calculated to be 0.998 (15).

FIG. 4.

Expression of A. marginale MSP2 protein during transmission feeding. Ticks were either transmission fed on calf 847 (F) or held at 26°C (26). Expression of MSP2 was examined at 6, 18, 42, and 72 h by using Western blots. MSP2 and MSP5 were detected as described for Fig. 2B. The positions of SGV1, SGV2, and MSP5 are indicated in the left margin, and molecular size markers are indicated in the right margin.

FIG. 5.

Presence of A. marginale in the salivary gland of acquisition-feeding ticks. (A) Full-length msp2 was detected by PCR in salivary glands obtained on days 2, 3, 5, 6, and 7 of the acquisition feed (A) on calf 873, day 7 of the holding period (H), and day 3 of the transmission feed (T) on calf 882. Blood was collected from calf 873 on day 3 during the acquisition feeding (blood). The reactions were carried out with primer pair MSP2:184-201 and MSP2:891-874. A reaction mixture without template served as negative control (H₂O). The position of the 700-bp fragment is indicated in the left margin. (B) Expression of MSP2 in salivary glands was examined before attachment of the ticks (0d) and day 7 (7d) of the holding period (H) after the acquisition feed on calf 873 by using Western blots. MSP2 and MSP5 were detected as described for Fig. 2B. The positions of SGV1, SGV2, and MSP5 are indicated in the left margin, and molecular size markers are indicated in the right margin.