Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.
Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.
Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.
The continuous human exploitation of environmental resources and the increase in human outdoor activities, which have allowed for the contact with arthropod vectors normally present in the field, has promoted the emergence and resurgence of vector-borne pathogens. Among these, Anaplasma phagocytophilum is an emerging bacterial pathogen transmitted to humans and other vertebrate hosts by ticks as they take a blood meal that causes human granulocytic anaplasmosis in the United States, Europe and Asia, with increasing numbers of affected people every year. Tick response to pathogen infection has been only partially characterized. In this study, global tissue-specific response and apoptosis signaling pathways were characterized in tick nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. The results demonstrated dramatic and complex tissue-specific response to A. phagocytophilum in the tick vector Ixodes scapularis, which reflected pathogen developmental cycle and the impact on tick apoptosis pathways. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and contributes information on tick-pathogen interactions and for development of novel control strategies for pathogen infection and transmission.
Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.
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.
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.
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.
Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.
Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species.
For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level.
These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.
Anaplasmosis; Genetics; Pig; Wild boar; Genomics; Immune response
The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis.
Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8±0.8×101 and 1.1±0.03×103 CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae.
This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the central United States.
The clinical findings, diagnostic tests, and treatment of clinical anemia in a mature Angus cow infected with the hemoplasma Mycoplasma wenyonii are described. Mycoplasma wenyonii has been previously reported to cause clinical anemia in young or splenectomized cattle; however, infection has not been associated with severe anemia in mature animals.
In 2009, we reported a novel form of delayed anaphylaxis to red meat, which is related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Most of these patients had tolerated meat for many years previously. The implication is that some exposure in adult life had stimulated the production of these IgE antibodies.
To investigate possible causes of this IgE antibody response, focusing on evidence related to tick bites, which are common in the region where these reactions occur.
Serum assays were carried out using biotinylated proteins and extracts bound to a streptavidin ImmunoCAP.
Prospective studies on IgE antibodies in three subjects following tick bites showed an increase in IgE to alpha-gal of twenty-fold or greater. Other evidence included i) a strong correlation between histories of tick bites and IgE to alpha-gal (χ2=26.8, p<0.001), ii) evidence that these IgE antibodies are common in areas where the tick Amblyomma americanum is common, and iii) a significant correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from A. americanum (rs=0.75, p<0.001).
The results presented here provide evidence that tick bites are a cause, or possibly the only cause, of IgE specific for alpha-gal in this area of the United States. Both the number of subjects becoming sensitized and the titer of IgE antibodies to alpha-gal are striking. Here we report the first example of a response to an ectoparasite giving rise to an important form of food allergy.
ticks; anaphylaxis; oligosaccharide; alpha-gal; IgE antibody to CCD
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.
Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection.
Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells.
Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.
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, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF).
Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis 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 D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism.
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.
The tick-borne pathogen Anaplasma marginale, which is endemic worldwide, is the type species of the genus Anaplasma (Rickettsiales: Anaplasmataceae). Rhipicephalus (Boophilus) microplus is the most important tick vector of A. marginale in tropical and subtropical regions of the world. Despite extensive characterization of the genetic diversity in A. marginale geographic strains using major surface protein sequences, little is known about the biogeography and evolution of A. marginale and other Anaplasma species. For A. marginale, MSP1a was shown to be involved in vector-pathogen and host-pathogen interactions and to have evolved under positive selection pressure. The MSP1a of A. marginale strains differs in molecular weight because of a variable number of tandem 23-31 amino acid repeats and has proven to be a stable marker of strain identity. While phylogenetic studies of MSP1a repeat sequences have shown evidence of A. marginale-tick co-evolution, these studies have not provided phylogeographic information on a global scale because of the high level of MSP1a genetic diversity among geographic strains.
In this study we showed that the phylogeography of A. marginale MSP1a sequences is associated with world ecological regions (ecoregions) resulting in different evolutionary pressures and thence MSP1a sequences. The results demonstrated that the MSP1a first (R1) and last (RL) repeats and microsatellite sequences were associated with world ecoregion clusters with specific and different environmental envelopes. The evolution of R1 repeat sequences was found to be under positive selection. It is hypothesized that the driving environmental factors regulating tick populations could act on the selection of different A. marginale MSP1a sequence lineages, associated to each ecoregion.
The results reported herein provided the first evidence that the evolution of A. marginale was linked to ecological traits affecting tick vector performance. These results suggested that some A. marginale strains have evolved under conditions that support pathogen biological transmission by R. microplus, under different ecological traits which affect performance of R. microplus populations. The evolution of other A. marginale strains may be linked to transmission by other tick species or to mechanical transmission in regions where R. microplus is currently eradicated. The information derived from this study is fundamental toward understanding the evolution of other vector-borne pathogens.
The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.
The impact of gene knockdown on A. marginale tick infections, both after acquiring infection and after a second transmission feeding, was determined and studied by light microscopy. Silencing of these genes had a different impact on A. marginale development in different tick tissues by affecting infection levels, the densities of colonies containing reticulated or dense forms and tissue morphology. Salivary gland infections were not seen in any of the gene-silenced ticks, raising the question of whether these ticks were able to transmit the pathogen.
The results of this RNAi and light microscopic analyses of tick tissues infected with A. marginale after the silencing of genes functionally important for pathogen development suggest a role for these molecules during pathogen life cycle in ticks.
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.
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.
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.
Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks.
Bovine anaplasmosis has been reported in several European countries, but the vector competency of tick species for Anaplasma marginale from these localities has not been determined. Because of the wide distributional range of Dermacentor reticulatus within Europe and the major role of Dermacentor spp. as a vector of A. marginale in the United States, we tested the vector competency of D. reticulatus for A. marginale.
Male D. reticulatus were allowed to feed for 7 days on a calf persistently infected with a Zaria isolate of A. marginale, after which they were removed and held off-host for 7 days. The ticks were then allowed to feed a second time for 7 days on a susceptible tick-naïve calf. Infection of calf No. 4291 was detected 20 days post exposure (p.i.) and confirmed by msp4 PCR. Thirty percent of the dissected acquisition fed ticks was infected. In addition, A. marginale colonies were detected by light microscopy in the salivary glands of the acquisition fed ticks. Transmission of A. marginale to calf No. 9191 was confirmed by examination of Giemsa-stained blood smears and msp4 PCR. Ticks were dissected after transmission feeding and presence of A. marginale was confirmed in 18.5% of the dissected ticks.
This study demonstrates that D. reticulatus males are competent vectors of A. marginale. Further studies are needed to confirm the vector competency of D. reticulatus for other A. marginale strains from geographic areas in Europe.
The tick-borne intracellular pathogen, Anaplasma phagocytophilum (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 A. phagocytophilum.
The human Sp110 and A. phagocytophilum msp4 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 A. phagocytophilum 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 A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of msp4 and normalizing against human Alu sequences.
While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels.
These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.
The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.
Bm86; Bm91; Subolesin; RNAi; Boophilus microplus; One-host tick
The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes.
Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously.
At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.
The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained from white-tailed deer in the United States. Sequence variation between strains of A. phagocytophilum (90 to 100% identity at the nucleotide level and 92 to 100% similarity at the protein level) was higher than in A. marginale. Phylogenetic analyses of msp4 sequences did not provide phylogeographic information but did differentiate strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs, and horses. The sequence analysis of the recently discovered A. phagocytophilum msp2 gene corroborated these results. The results reported here suggest that although A. phagocytophilum-like organisms from white-tailed deer may be closely related to A. phagocytophilum, they could be more diverse. These results suggest that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans.
The rickettsial pathogen Anaplasma marginale assembles an actin filament bundle during intracellular infection. Unlike other bacterial pathogens that generate actin filament tails, A. marginale infects mature erythrocytes, and the F-actin appendages are assembled on the cytoplasmic surface of a vacuole containing several organisms. To identify A. marginale molecules associated with these filaments, two complementary approaches were used: matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tandem mass spectrometry of A. marginale proteins identified with an appendage-specific monoclonal antibody and expression screening of an A. marginale phage library. Amino acid and nucleotide sequences were mapped to a full-length gene in the genome of the St. Maries strain of A. marginale; the correct identification was confirmed by expression of full-length recombinant protein and its reactivity with appendage-specific antibodies. Interestingly, there is marked variation in the abilities of diverse A. marginale strains to assemble the F-actin appendages. Comparison of four strains, the Florida, Illinois, St. Maries, and Virginia strains, revealed substantial polymorphism in the gene encoding the appendage-associated protein, with amino acid sequence identity of as low as 34% among strains. However, this variation does not underlie the differences in expression, as there is no specific polymorphism associated with loss of ability to assemble actin appendages. In contrast, the ability to assemble an actin filament bundle reflected dramatic strain-specific differences in the expression level of the appendage-associated protein. Understanding how this protein influences the cycle of invasion, replication, and egress in the host cell may provide new insights into pathogen-host interactions.
Bovine anaplasmosis is a vector-borne disease that results in substantial economic losses in other parts of the world but so far not in northern Europe. In August 2002, a fatal disease outbreak was reported in a large dairy herd in the Swiss canton of Grisons. Diseased animals experienced fever, anorexia, agalactia, and depression. Anemia, ectoparasite infestation, and, occasionally, hemoglobinuria were observed. To determine the roles of vector-borne pathogens and to characterize the disease, blood samples were collected from all 286 animals: 50% of the cows were anemic. Upon microscopic examination of red blood cells, Anaplasma marginale inclusion bodies were found in 47% of the cows. The infection was confirmed serologically and by molecular methods. Interestingly, we also found evidence of infections with Anaplasma phagocytophilum, large Babesia and Theileria spp., and Mycoplasma wenyonii. The last two species had not previously been described in Switzerland. Anemia was significantly associated with the presence of the infectious agents detected, with the exception of A. phagocytophilum. Remarkably, concurrent infections with up to five infectious vector-borne agents were detected in 90% of the ill animals tested by PCR. We concluded that A. marginale was the major cause of the hemolytic anemia, while coinfections with other agents exacerbated the disease. This was the first severe disease outbreak associated with concurrent infections with vector-borne pathogens in alpine Switzerland; it was presumably curtailed by culling of the entire herd. It remains to be seen whether similar disease outbreaks will have to be anticipated in northern Europe in the future.
Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne rickettsial pathogen of cattle that multiplies in erythrocytes and tick cells. Major surface protein 1a (MSP1a) and MSP1b form the MSP1 complex of A. marginale, which is involved in adhesion of the pathogen to host cells. In this study we tested the hypothesis that MSP1a and MSP1b were glycosylated, because the observed molecular weights of both proteins were greater than the deduced molecular masses. We further hypothesized that the glycosylation of MSP1a plays a role in adhesion of A. marginale to tick cells. Native and Escherichia coli-derived recombinant MSP1a and MSP1b proteins were shown by gas chromatography to be glycosylated and to contain neutral sugars. Glycosylation of MSP1a appeared to be mainly O-linked to Ser/Thr residues in the N-terminal repeated peptides. Glycosylation may play a role in adhesion of A. marginale to tick cells because chemical deglycosylation of MSP1a significantly reduced its adhesive properties. Although the MSP1a polypeptide backbone alone was adherent to tick cell extract, the glycans in the N-terminal repeats appeared to enhance binding and may cooperatively interact with one or more surface molecules on host cells. These results demonstrated that MSP1a and MSP1b are glycosylated and suggest that the glycosylation of MSP1a plays a role in the adhesion of A. marginale to tick cells.