The fine structure of developmental stages of Babesia caballi in the salivary glands of Hyalomma truncatum

The development of Babesia caballi in the salivary glands of Hyalomma truncatum was studied at the electron microscopic level. Kinetes were first observed in the salivary glands of ticks on Day 2 of tick feeding and on each subsequent day of feeding until engorgement on Day 8. Sporogony appeared to involve the formation of cytomeres. After continued nuclear division, sporozoites formed when individual rounded nuclei were incorporated into portions of cytoplasm. Sporozoites were first observed on Day 4 of tick feeding and contained typical Babesia spp. organelles with a polar ring and up to 4 rhoptries, spherical bodies, a nucleus, mitochondria, endoplasmic reticulum and micronemes. The infection rate in the ticks was approximately 80%.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.lmchunu2014mn201

Comparative infection rates of Theileria parva lawrencei in salivary glands of Rhipicephalus appendiculatus and Rhipicephalus zambeziensis

Three cattle, which had been experimentally infected with Theileria parva lawrencei and maintained as carriers of the infection, were each infested simultaneously with clean nymphal Rhipicephalus appendiculatus and Rhipicephalus zambeziensis in ear bags on separate ears. After moulting, the ensuing adult ticks were fed on rabbits for 4 days and their salivary glands were examined for infective stages of the parasite. Microscopic examination revealed significantly higher infection rates in the salivary glands of R. zambeziensis than in R. appendiculatus which may indicate an increased vector efficiency of R. zambeziensis for T. p. lawrencei.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.lmchunu2014mn201

An ultrastructural study of the development of Babesia occultans in the salivary glands of adult Hyalomma marginatum rufipes

The development of Babesia occultans in the salivary glands of adult Hyalomma marginatum rufipes was studied with the electron microscope. Sporogony involved a process of multiple fission in which sporozoites formed from the periphery of a polymorphous sporont. Different stages of development were found concurrently in individual acini as well as within individual acinar cells. Mature sporozoites were found on Day 3 post-tick attachment and measures 3,0-3,5 x 1,5 µm. The apical complex consisted of a polar ring and 4-6 rhoptries. Micronemes were concentrated anteriorly and 1 or 2 spherical bodies were identlfied in each sporozoite. The general pattern of development was similar to that described in several other Babesia spp. but distinct morphologic differences were noted.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.lmchunu2014mn201

Anaplasma phagocytophilum and Anaplasma marginale Elicit Different Gene Expression Responses in Cultured Tick Cells

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells

Reciprocal regulation of NF-kB (Relish) and subolesin in the tick vector, Ixodes scapularis

[Background]: Tick Subolesin and its ortholog in insects and vertebrates, Akirin, have been suggested to play a role in the immune response through regulation of nuclear factor-kappa B (NF-kB)-dependent and independent gene expression via interaction with intermediate proteins that interact with NF-kB and other regulatory proteins, bind DNA or remodel chromatin to regulate gene expression. The objective of this study was to characterize the structure and regulation of subolesin in Ixodes scapularis. I. scapularis is a vector of emerging pathogens such as Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti that cause in humans Lyme disease, anaplasmosis and babesiosis, respectively. The genome of I. scapularis was recently sequenced, and this tick serves as a model organism for the study of vector-host-pathogen interactions. However, basic biological questions such as gene organization and regulation are largely unknown in ticks and other arthropod vectors. [Principal Findings]: The results presented here provide evidence that subolesin/akirin are evolutionarily conserved at several levels (primary sequence, gene organization and function), thus supporting their crucial biological function in metazoans. These results showed that NF-kB (Relish) is involved in the regulation of subolesin expression in ticks, suggesting that as in other organisms, different NF-kB integral subunits and/or unknown interacting proteins regulate the specificity of the NF-kB-mediated gene expression. These results suggested a regulatory network involving cross-regulation between NF-kB (Relish) and Subolesin and Subolesin auto-regulation with possible implications in tick immune response to bacterial infection. [Significance]: These results advance our understanding of gene organization and regulation in I. scapularis and have important implications for arthropod vectors genetics and immunology highlighting the possible role of NF-kB and Subolesin/Akirin in vector-pathogen interactions and for designing new strategies for the control of vector infestations and pathogen transmission.This research was supported by grants BFU2008-01244/BMC and BFU2011-23896 to JF, the Oklahoma Agricultural Experimental Grant 1669 and the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK, and the EU FP7 ANTIGONE project number 278976. V. Naranjo was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007-2013), Spain. N. Ayllón was funded by Ministerio de Educación y Ciencia (MEC), Spain.Peer Reviewe

Expression of Heat Shock and Other Stress Response Proteins in Ticks and Cultured Tick Cells in Response to Anaplasma spp. Infection and Heat Shock

Ticks are ectoparasites of animals and humans that serve as vectors of Anaplasma and other pathogens that affect humans and animals worldwide. Ticks and the pathogens that they transmit have coevolved molecular interactions involving genetic traits of both the tick and the pathogen that mediate their development and survival. In this paper, the expression of heat shock proteins (HSPs) and other stress response proteins (SRPs) was characterized in ticks and cultured tick cells by proteomics and transcriptomics analyses in response to Anaplasma spp. infection and heat shock. The results of these studies demonstrated that the stress response was activated in ticks and cultured tick cells after Anaplasma spp. infection and heat shock. However, in the natural vector-pathogen relationship, HSPs and other SRPs were not strongly activated, which likely resulted from tick-pathogen coevolution. These results also demonstrated pathogen- and tick-specific differences in the expression of HSPs and other SRPs in ticks and cultured tick cells infected with Anaplasma spp. and suggested the existence of post-transcriptional mechanisms induced by Anaplasma spp. to control tick response to infection. These results illustrated the complexity of the stress response in ticks and suggested a function for the HSPs and other SRPs during Anaplasma spp. infection

Subolesin expression in response to pathogen infection in ticks

<p>Abstract</p> <p>Background</p> <p>Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, <it>Anaplasma marginale</it>. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with <it>Anaplasma</it>, <it>Ehrlichia</it>, <it>Rickettsia</it>, <it>Babesia </it>or <it>Theileria </it>spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in <it>Dermacentor variabilis </it>males exposed to various pathogens by capillary feeding (CF).</p> <p>Results</p> <p>Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with <it>A. marginale</it>. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in <it>D. variabilis </it>infected with <it>A. marginale </it>and other tick-borne pathogens resulted in lower infection levels, while infection with <it>Francisella tularensis </it>increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in <it>D. variabilis </it>exposed to <it>Escherichia coli</it>, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism.</p> <p>Conclusions</p> <p>Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.</p

Genetic diversity of Anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes several pathogens of veterinary and human medical importance. An understanding of the diversity of Anaplasma major surface proteins (MSPs), including those MSPs that modulate infection, development of persistent infections, and transmission of pathogens by ticks, is derived in part, by characterization and phylogenetic analyses of geographic strains. Information concerning the genetic diversity of Anaplasma spp. MSPs will likely influence the development of serodiagnostic assays and vaccine strategies for the control of anaplasmosi

Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells

<p>Abstract</p> <p>Background</p> <p>The tick-borne intracellular pathogen, <it>Anaplasma phagocytophilum </it>(Rickettsiales: Anaplasmataceae) causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB) components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with <it>A. phagocytophilum</it>.</p> <p>Methods</p> <p>The human Sp110 and <it>A. phagocytophilum msp4 </it>mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on <it>A. phagocytophilum </it>infection was determined by RNA interference (RNAi). The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA). The <it>A. phagocytophilum </it>infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of <it>msp4 </it>and normalizing against human <it>Alu </it>sequences.</p> <p>Results</p> <p>While Sp110 mRNA levels increased concurrently with <it>A. phagocytophilum </it>infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels.</p> <p>Conclusion</p> <p>These results demonstrated that Sp110 expression is required for <it>A. phagocytophilum </it>infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which <it>A. phagocytophilum </it>modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.</p

Evidence of the role of tick subolesin in gene expression

Background: Subolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression.Results: Two subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites.Conclusion: Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks.Peer reviewedVeterinary PathobiologyBiochemistry and Molecular Biolog