Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus.

BACKGROUND: Babesia bovis, an intra-erythrocytic apicomplexan parasite, is one of the causative agents of bovine babesiosis, the most important tick-borne disease of cattle in tropical and subtropical regions. Babesia bovis has a complex life-cycle that includes sexual development within the tick vector. The development of a transmission blocking vaccine to control bovine babesiosis requires the identification of antigens displayed on the surface of the parasite during its development within tick vectors. Four B. bovis cysteine-rich GCC2/GCC3 domain protein (BboGDP) family members were previously identified and are differentially expressed as discrete pairs by either blood stages or kinetes. In this study we focused on two family members, BboGDP1 and -3, that are expressed by Babesia parasites during tick infection. METHODS AND RESULTS: Transcription analysis using quantitative PCR demonstrated that BboGDP1 and -3 were upregulated in in vitro-induced sexual stage parasites and during parasite development in the tick midgut. Moreover, protein expression analysis of BboGDP1 and -3 during the development of sexual stages in in vitro culture was consistent with their transcription profile. Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of BboGDP1 and -3 on in vitro-induced sexual stage parasites. In addition, fixed immunofluorescence analysis showed reactivity of anti-BboGDP1 and -3 polyclonal antibodies to kinetes. CONCLUSIONS: The collective data indicate that BboGDP1 and -3 are expressed by kinetes and on the surface of sexual stages of the parasites. The identified parasite surface membrane proteins BboGDP1 and -3 are potential candidates for the development of a B. bovis transmission blocking vaccine.

The transcription factor Relish controls Anaplasma marginale infection in the bovine tick Rhipicephalus microplus.

Rhipicephalus microplus is an important biological vector of Anaplasma marginale, the etiological agent of bovine anaplasmosis. The knowledge of tick immune responses to control bacterial infections remains limited. In this study, we demonstrate that transcription factor Relish from the IMD signaling pathway has an important role in the control of A. marginale infection in ticks. We found that RNA-mediated silencing of Relish caused a significant increase in the number of A. marginale in the midgut and salivary glands of R. microplus. In addition, the IMD pathway regulates the expression of the gene that encodes the antimicrobial peptide (AMP) microplusin. Moreover, microplusin expression was up-regulated in the midgut (2×) and salivary glands (8×) of A. marginale infected R. microplus. Therefore, it is plausible to hypothesize that microplusin may be involved in the A. marginale control. This study provides the first evidence of IMD signaling pathway participation on the A. marginale control in R. microplus.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

A divergent Artiodactyl MYADM-like repeat is associated with erythrocyte traits and weight of lamb weaned in domestic sheep.

A genome-wide association study (GWAS) was performed to investigate seven red blood cell (RBC) phenotypes in over 500 domestic sheep (Ovis aries) from three breeds (Columbia, Polypay, and Rambouillet). A single nucleotide polymorphism (SNP) showed genome-wide significant association with increased mean corpuscular hemoglobin concentration (MCHC, P = 6.2×10(-14)) and genome-wide suggestive association with decreased mean corpuscular volume (MCV, P = 2.5×10(-6)). The ovine HapMap project found the same genomic region and the same peak SNP has been under extreme historical selective pressure, demonstrating the importance of this region for survival, reproduction, and/or artificially selected traits. We observed a large (>50 kb) variant haplotype sequence containing a full-length divergent artiodactyl MYADM-like repeat in strong linkage disequilibrium with the associated SNP. MYADM gene family members play roles in membrane organization and formation in myeloid cells. However, to our knowledge, no member of the MYADM gene family has been identified in development of morphologically variant RBCs. The specific RBC differences may be indicative of alterations in morphology. Additionally, erythrocytes with altered morphological structure often exhibit increased structural fragility, leading to increased RBC turnover and energy expenditure. The divergent artiodactyl MYADM-like repeat was also associated with increased ewe lifetime kilograms of lamb weaned (P = 2×10(-4)). This suggests selection for normal RBCs might increase lamb weights, although further validation is required before implementation in marker-assisted selection. These results provide clues to explain the strong selection on the artiodactyl MYADM-like repeat locus in sheep, and suggest MYADM family members may be important for RBC morphology in other mammals.