In the present study we identified R. microplus male salivary gland genes differentially expressed in response to A. marginale infection by use of SSH and real-time RT-PCR. Development and multiplication of A. marginale in salivary gland cells involves molecular interactions between pathogen- and tick-derived molecules. Salivary gland, the tissue of interest in this study, is a critical site in the developmental cycle from where the pathogen is transmitted to cattle. Recently, tick salivary gland proteins were shown to play a role in the infection and transmission of Borrelia burgdorferi [16, 17], A. phagocytophilum  and A. marginale . A. marginale membrane surface proteins involved in tick salivary gland colonization have been identified and partially characterized [20, 21]. Understanding the molecular mechanisms of A. marginale -tick interactions for R. microplus, one of the most important vectors of A. marginale worldwide, is fundamental toward development of novel control measures .
Some of the genes identified by SSH, including those genes encoding for putative tick cement proteins, female specific histamine binding protein, IgG binding protein C, salivary gland-associated protein 64P, flagelliform silk protein and von Willebrand factor, were identified previously in different tick species and appear to be involved in tick feeding or pathogen infection [10, 23–25]. However, most of the differentially expressed genes identified in this study have not been shown to be associated with tick-pathogen interaction previously. Some cellular functions affected by A. marginale infection of R. microplus, such as cell structure and enzymatic processes, were reported previously in infected tick IDE8 cultured cells . The discrepancy observed for some studied genes between SSH and real-time RT-PCR results may reflect differences between both methods for identifying differentially expressed genes or the presence of multiple sequences targeted during RT-PCR reactions that affect the results of mRNA quantification for some genes.
In a recent study, genes differentially expressed in cultured IDE8 tick cells in response to A. marginale infection were identified and functional studies conducted in D. variabilis suggested that these genes may play different roles during pathogen infection, development and trafficking from midguts to salivary glands . Some of the genes identified by de la Fuente et al.  such as gluthathione S-tranferase, selenoprotein M and ferritin were also shown to be differentially expressed in R. microplus salivary glands in response to A. marginale infection. However, these genes were absent from the current EST dataset which could be due to differences in the system used for EST discovery (cultured IDE8 tick cells versus R. microplus salivary glands) and/or other factors such as tick species and/or A. marginale strain and infection levels.
While tick cell lines have been used successfully in A. marginale functional genomics studies , this is the first report of the use of the BME26 tick cell line derived originally from a natural vector of A. marginale for functional studies of tick-pathogen interactions. Since these studies were conducted on ticks and tick cells of the same species, most of the genes identified in tick salivary glands were also amplified from cultured BME26 tick cells. However, expression profiles of selected genes observed in cultured BME26 cells were not identical to that found in tick salivary glands. For example, the expression of the putative von Willebrand factor (94Will) was down-regulated in tick salivary glands but up-regulated in cultured BME26 tick cells infected with A. marginale. These differences may have resulted from tissue-specific regulation of gene expression or because we only observed early stages of infection in the cultured BME26 tick cells (6-72 hpi). As reported previously , results of studies using cultured tick cells must be validated in naturally infected ticks. Interestingly, expression of putative vacuolar H+-ATPase (36vATP) was significantly up-regulated in A. marginale -infected cultured BME26 cells, as reported for previous gene expression studies of cultured IDE8 cells in response to A. marginale infection .
RNAi was used in this study to assign the effect of selected gene knockdown on A. marginale infection and multiplication in ticks. Although statistically significant for flagelliform silk protein (100Silk) only, results of RNAi experiments suggested that putative von Willebrand factor (94Will), flagelliform silk protein (100Silk) and subolesin could play a role in pathogen infection of R. microplus male salivary glands. RNAi experiments in cultured BME26 tick cells provided further evidence that flagelliform silk protein (100Silk) and subolesin may play a role in A. marginale infection and/or multiplication in tick cells and suggested that metallothionein (93 Meth) may be involved tick defense against pathogen infection.
The flagelliform silk protein was identified previously in tick and orb weaving spider salivary glands but its function was not linked to pathogen infection [26–28]. Mulenga et al.  demonstrated that the flagelliform silk protein may be involved in tick attachment. In previous studies of I. ricinus after B. burgdorferi infection, the von Willebrand factor was isolated from tick salivary glands and shown to be up-regulated but its possible role in infection was not studied . A von Willebrand factor-like motif is present in the major hemelipoglycoprotein found in ixodid ticks and this protein has been shown to play a role as a heme-sequestering factor during tick feeding . Therefore, silencing of these genes may affect tick feeding, mortality and development of A. marginale in salivary glands. However, as shown previously for subolesin , gene expression studies in cultured BME26 tick cells have provided evidence that that the flagelliform silk protein may play a role in the infection of ticks with A. marginale.
The results for gene expression and silencing of subolesin in R. microplus male salivary glands and cultured BME26 cells infected with A. marginale reported herein are in agreement with previous studies in which subolesin knockdown reduced A. marginale infection in D. variabilis and cultured IDE8 cells [11, 19]. Subolesin, discovered as a tick protective antigen in I. scapularis, has been shown to be conserved in many tick species [31, 32]. Subolesin was shown by both RNAi gene knockdown and immunization trials using the recombinant protein to protect vertebrate hosts against tick infestations, reduce tick survival and reproduction, and cause degeneration of gut, salivary gland, reproductive tissues and embryos [31–37]. Targeting of subolesin by RNAi or vaccination also decreased tick vector capacity for A. marginale and A. phagocytophilum . In addition, subolesin was shown to function in the control of gene expression in ticks [38, 39] and to be differentially expressed in Anaplasma -infected ticks and cultures tick cells [11, 40]. However, subolesin expression in R. microplus tick salivary glands and cultured BME26 cells was different to previous reports showing up-regulation in A. marginale -infected D. variabilis salivary glands and cultured IDE8 cells . These differences could be due to tick species-specific differences in gene regulation or to other factors such as pathogen strain and infection levels. Nonetheless, these results expanded our knowledge on the role of subolesin in tick- Anaplasma interactions.
Metallothioneins are a family of low molecular weight proteins with a high affinity for divalent metals that function in cell detoxification, apoptosis, stress response and immunity [41–43]. Metallothioneins control the cellular zinc ion levels, which are known to be important in the immune system, and their expression has been associated with protective response against pathogens [44–48]. The results suggested a role for tick metallothioneins in defense against bacterial infections. Interestingly, selenoproteins that regulate the levels of another important trace mineral in the organism were suggested to participate in the cellular response to limit A. marginale infection in tick cells .
Although dsRNA sequences used in this study do not contain any significant overlap with other known R. microplus genes, the possibility of off-target gene silencing effects cannot be excluded due to the limited amount of sequence data available. However, RNAi seems to be very sequence-specific in ticks with little off-target effects . Availability of the complete R. microplus genome sequence data will facilitate screening for potential off-target effects. These can subsequently be minimized by avoiding the use of dsRNAs or siRNAs containing sequences which are present in multiple genes.
In our study R. microplus male salivary gland genes differentially expressed in response to A. marginale infection were identified by using SSH approach. Recently a R. microplus microarray (NimbleGen) has been developed and used for the analysis of acaricide- inducible genes in R. microplus . Microarray chip hybridization could be an alternative approach for identifying R. microplus differentially expressed genes in response to A. marginale infection.