Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission. A Roche NimbleGen high-density gene expression microarray was custom designed based on the expressed sequence tag (EST) database, B. microplus Gene Index Version 2 (BmiGI V2) for R. microplus. The expression level of 14,447 R. microplus genes was analyzed from total RNA extracted from 10 different tick tissue samples; 30 arrays were included since triplicates of each different sample were analyzed as follow: unfed (midgut and salivary glands), blood feeding (2 days midgut and 2, 6 and 9 days salivary glands), A. marginale-infected blood feeding (2 days midgut and 2, 6 and 9 days salivary glands).

Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission.

Project description:Ticks are blood feeding arthropod ectoparasites that transmit pathogens, which cause diseases in humans and animals worldwide. In the past ten decades, the continuous human exploitation of environmental resources and the increase in human outdoor activities has promoted contact with arthropod vectors normally present in the wild, resulting in increased transmission of vector-borne pathogens. In addition, vector populations are expanding in response to climate change and human interventions that impact reservoir host movement and human exposure to infected vectors. Among these emerging vector-borne pathogens, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) has become an important tick-borne pathogen in the United States, Europe and Asia, with increasing numbers of infected people and animals every year. Diseases caused by A. phagocytophilum include human granulocytic anaplasmosis (HGA), equine and canine granulocytic anaplasmosis and tick-borne fever (TBF) in ruminants. The natural infection cycle of A. phagocytophilum is dependent upon the presence of infected vertebrate reservoir hosts and Ixodid tick vectors. In the United States and Europe the main vector species are Ixodes scapularis, Ixodes pacificus, and Ixodes ricinus, while a wide range of mammals, lizards, and birds serve as reservoir hosts for various A. phagocytophilum genotypes. A. phagocytophilum initially infects tick midgut cells and then subsequently develops in salivary glands for transmission to susceptible hosts during tick feeding where the pathogen infects granulocytic cells, primarily neutrophils. Anaplasma phagocytophilum develops within membrane-bound inclusions in the host cell cytoplasm. This pathogen has evolved with its tick and vertebrate hosts through dynamic processes involving genetic traits of the pathogen and hosts that collectively mediate pathogen infection, development, persistence, and survival. However, the mechanisms used by A. phagocytophilum for molecular mechanisms involved in tick-pathogen interactions have not been fully characterized. The objective of this study is to characterize the dynamics of the microRNA response in the tick vector Ixodes scapularis in response to A. phagocytophilum infection. To address this objective, the composition of tick microRNAs was characterize using RNA sequencing in I. scapularis tick cells in response to A. phagocytophilum infection. The discovery of these mechanisms provides evidence that a control strategy could be developed targeted at both vertebrate and tick hosts for more complete control of A. phagocytophilum and its associated diseases.

Project description:Studying the salivary glands potential role during intestinal infections,using advantage from the ingenious GAL4/UAS-system available in the fly Epithelial immunity is a very simple but nevertheless indispensable type of immune response. Amongst all epithelial tissues, the intestine and the airways are outstanding, because they provide huge surface areas, where colonization or invasion of potential pathogens has to be obviated. The intestine is of special interest, because it has to hold the balance between tolerance and immunity, to protect the own microbial flora. An essential part of the intestinal tract has been completely overlooked, the salivary glands. They are the gatekeepers of the intestinal system, being essential for various aspects of the intestineâs immunity. Using the bipartite Gal4/UAS expression system, it is possible to activate the immune system ectopically in different organs of the fly. Activation of the IMD-pathway in the salivary glands is possible; because a very specific driver line is available that directs expression of any gene of interest into the salivary glands only. Flies, where the IMD-pathway in the salivary glands has been activated have a very distinct phenotype. They show a smaller body length as well as a smaller salivary gland length than the parental animals Microarray data analysis showed that 457 genes were upregulated and 578 genes downregulated. Interestingly, the sets of regulated genes show only a very small overlap with the canonical set of Drosophila immune genes. Other physiological scenarios such as autophagic cell death are apparently also not activated upon IMD-pathway activation. Among the regulated genes, those that code for signaling associated protease activity are significantly modulated. This holds especially true for presenilin and the signal peptide peptidase. The comparison of the transcriptional events induced following IMD-activation in the trachea and the salivary glands shows also only a small overlap, indicating that the general IMD-activated core transcriptome is rather small. In conclusion, the salivary glands may a very good tool to study the physiological role of selected genes that are of importance for human health such as the Alzheimerâs disease related presenilin gene in a functional environment. Ectopic activiation of the Immune deficiency pathway in the larval salivary glands using the Gal4/UAS-system of Brand and Perrimon. The activation was induced by the overexpression of the pattern recognition receptor PGRP-LCx using a driver line which was specific for the salivary glands.In general four replicates were performed including dye-swaps in two-colour arrays.

Project description:Ticks are obligate blood feeding ectoparasites that transmit a wide variety of pathogenic microorganisms to their vertebrate hosts. Amblyomma sculptum is vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, the most lethal rickettsiosis that affects humans. It is known that the transmission of pathogens by ticks is mainly associated with the physiology of the feeding process. Pathogens that are acquired with the blood meal must first colonize the tick gut and later the salivary glands in order to be transmitted during a subsequent blood feeding via saliva. Tick saliva contains a complex mixture of bioactive molecules with anticlotting, antiplatelet aggregation, vasodilatory, anti-inflammatory, and immunomodulatory properties to counteract both the hemostasis and defense mechanisms of the host. Besides facilitating tick feeding, the properties of saliva may also benefits survival and establishment of pathogens in the host. In the current study, we compared the sialotranscriptome of unfed A. sculptum ticks and those fed for 72 h on rabbits using next generation RNA sequencing (RNA-seq). The total of reads obtained were assembled in 9,560 coding sequences (CDSs) distributed in different functional classes. CDSs encoding secreted proteins, including lipocalins, mucins, protease inhibitors, glycine-rich proteins, metalloproteases, 8.9 kDa superfamily members, and immunity-related proteins were mostly upregulated by blood feeding. Selected CDSs were analyzed by real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR), corroborating the transcriptional profile obtained by RNA-seq. Finally, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed 124 proteins in saliva of ticks fed for 96-120 h. The corresponding CDSs of 59 of these proteins were upregulated in salivary glands of fed ticks. To the best of our knowledge, this is the first report on the proteome of A. sculptum saliva. The functional characterization of the identified proteins might reveal potential targets to develop vaccines for tick control and/or blocking of R. rickettsii transmission as well as pharmacological bioproducts with antihemostatic, anti-inflammatory and antibacterial activities.

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