Transcriptomics data integration reveals Jak-STAT as a common pathway affected by pathogenic intracellular bacteria in natural reservoir hosts

The study of the host-pathogen interface in natural reservoir hosts is essential to identify host-cell mechanisms affected by bacterial infection and persistence. Herein we used the Database for Annotation, Visualization and Integrated Discovery (DAVID) to integrate transcriptomics data and find common host-cell biological processes, molecular functions and pathways affected by pathogenic intracellular bacteria of the genera Anaplasma, Brucella and Mycobacterium during infection and persistence in two natural reservoir hosts, wild boar and sheep. The results showed that the upregulation of host innate immune pro-inflammatory genes and signaling pathways constitutes a general antibacterial mechanism in response to intracellular bacteria. Pathway focused analysis revealed a role for the Jak-STAT pathway during bacterial intracellular infection, a fact reported before in Mycobacterium infected cells but not during Brucella spp. and A. phagocytophilum infection. A clear activation of the Jak-STAT pathway was observed in A. phagocytophilum infected wild boar and sheep when compared to uninfected controls. Brucella spp. infection resulted in a balanced regulation of the Jak-STAT signaling and M. bovis infection of wild boar clearly produced a downregulation of some of the Jak-STAT effectors such as IL5 and TKY2. These results suggested that mycobacteria and brucellae induce host innate immune responses while manipulating adaptive immunity to circumvent host-cell defenses and establish infection. In contrast, A. phagocytophilum infection induces both innate and adaptive immunity, those suggesting that this pathogen uses other mechanisms to circumvent host-cell defenses by downregulating other adaptive immune genes and delaying the apoptotic death of neutrophils through activation of the Jak-STAT pathway among other mechanisms.This research was supported by the Grupo Santander and Fundación Marcelino Botín, Spain (project Control of Tuberculosis in Wildlife) and the EU FP7, ANTIGONE project number 278976. R.C. Galindo was funded by Ministerio de Ciencia y Educación (MEC), Spain.Peer Reviewe

IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.XYL was supported by funds from the China Scholarship Council (CSC). This work was funded by INRA and EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext037 (http://www.edenext.eu). This work was also partially supported by the Spanish Secretaría de Estado de Investigación, Desarrollo e Innovación, Ministerio de Economía y Competitividad project BFU2011-23896.Peer Reviewe

Molecular identification of Cordylobia anthropophaga Blanchard (Diptera: Calliphoridae) larvae collected from dogs (Canis familiaris) in Jos South, Plateau State, Nigeria

This work is licensed under the Creative Commons Attribution License.-- et al.Myiasis-causing larvae were extracted from dogs attending veterinary clinics in Plateau State, Nigeria and subjected to molecular analysis involving polymerase chain reaction amplification of the 28S rRNA gene of blowflies, cloning and sequencing techniques. All larvae were confirmed as Cordylobia anthropophaga Blanchard (Diptera: Calliphoridae) after the initial morphological identification. This is the first molecular identification of any myiasis-causing fly species in Nigeria and may serve as a reliable alternative to morphological identification where samples are not well preserved or difficult to identify to species level.We thank at the Instituto de Investigacion en Recursos Cinegiticos, IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain for providing the facilities to carry out this research.Peer Reviewe

Progress in the control of bovine tuberculosis in Spanish wildlife

Special issue: 5th International Conference on Mycobacterium bovis.-- HAL Id: hal-00701898Despite the compulsory test and slaughter campaigns in cattle, bovine tuberculosis (bTB) is still present in Spain, and the role of wildlife reservoirs is increasingly recognized. We provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination.At the high densities and in the particular circumstances of Mediterranean environments, wild ungulates, mainly Eurasian wild boar and red deer, are able to maintain Mycobacterium bovis circulation even in absence of domestic livestock. Infection is widespread among wild ungulates in the south of the country, local infection prevalence being as high as 52% in wild boar and 27% in red deer. Risk factors identified include host genetic susceptibility, abundance, spatial aggregation at feeders and waterholes, scavenging, and social behaviour. An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Southwestern Spain. Sporadic cases of badger TB have been detected, further complicating the picture.Gene expression profiles were characterized in European wild boar and Iberian red deer naturally infected with M. bovis. The comparative analysis of gene expression profiles in wildlife hosts in response to infection advanced our understanding of the molecular mechanisms of infection and pathogenesis, revealed common and distinctive host responses to infection and identified candidate genes associated with resistance to bTB and for the characterization of host response to infection and vaccination.Ongoing research is producing valuable knowledge on vaccine delivery, safety and efficacy issues. Baits for the oral delivery of BCG vaccine preparations to wild boar piglets were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy. Safety experiments yielded no isolation of M. bovis BCG from faeces, internal organs at necropsy and the environment, even after oral delivery of very high doses. Finally, preliminary vaccination and challenge experiments suggested that a single oral BCG vaccination may protect wild boar from infection by a virulent M. bovis field strain.Studies on TB at IREC are supported by Grupo Santander – Fundacion Marcelino Botin, and by TB-STEP EU FP7 212414, INIA-MICINN FAU 06-017, and PAI07-0062-6611 Castilla – La Mancha.Peer Reviewe

Nuclease Tudor-SN is involved in tick dsRNA-mediated RNA interference and feeding but not in defense against flaviviral or anaplasma phagocytophilum rickettsial infection

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al.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.This research was supported by grants BFU2011-23896 and the European Union FP7 ANTIGONE project number 278976 (JdlF). NA was funded by Ministerio de Educacion y Ciencia, Spain. VN was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007–2013), Spain. RS was supported by the project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and by the Grant 13-12816P (GA CR). OH was supported by the Grant Agency of the Czech Republic grant no. 13-27630P and European Union FP7 MODBIOLIN project number 316304. CR and LBS were supported by the United Kingdom Biotechnology and Biological Sciences Research Council's National Capability Grant to the Pirbright Institute.Peer Reviewe

A systems biology approach to the characterization of stress response in Dermacentor reticulatus tick unfed larvae

[Background]: Dermacentor reticulatus (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in D. reticulatus unfed larvae, with special emphasis on stress response. [Principal Findings]: The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as D. reticulatus with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in D. reticulatus unfed larvae and a Rickettsia sp. similar to R. raoultii was identified in these ticks. [Significance]: The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in D. reticulatus ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens.This research was supported by grants BFU2011-23896 and the EU FP7 ANTIGONE project number 278976. M. Popara is an Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). N. Ayllón and R.C. Galindo were funded by MEC, Spain.Peer Reviewe

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

Systems Biology of Tissue-Specific Response to Anaplasma phagocytophilum Reveals Differentiated Apoptosis in the Tick Vector Ixodes scapularis

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.This research was supported by grants BFU2011-23896, the EU FP7 ANTIGONE project number 278976, the Oklahoma Agricultural Experiment Grant 1669 and the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK. NA and RCG were funded by MEC, Spain. RS was supported by the project Postdok\_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and the Grant 13-12816P (GA CR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Experimental infection of Eurasian wild boar with Mycobacterium avium subsp. avium

The Eurasian wild boar (Sus scrofa) is increasingly relevant as a host for several pathogenic mycobacteria. We aimed to characterize the first experimental Mycobacterium avium subsp. avium (MAA) infection in wild boar in order to describe the lesions and the immune response as compared to uninfected controls. Twelve 1-4-month-old wild boar piglets were housed in class III bio-containment facilities. Four concentrations of MAA suspension were used: 10, 102 and 104 mycobacteria (2 animals each, oropharyngeal route) and 2.5×106 mycobacteria (2 animals each by the oropharyngeal and nasal routes). No clinical signs were observed and pathology evidenced a low pathogenicity of this MAA strain for this particular host. Bacteriological and pathological evidence of successful infection after experimental inoculation was found for the group challenged with 2.5×106 mycobacteria. These four wild boar showed a positive IFN-γ response to the avian PPD and the real-time RT-PCR data revealed that three genes, complement component C3, IFN-γ and RANTES, were significantly down regulated in infected animals. These results were similar to those found in naturally and experimentally M. bovis-infected wild boar and may constitute biomarkers of mycobacterial infection in this species.The study was funded by INIA-MICINN research grant FAU2006-00017 and Plan NacionalAGL2008-03875. Studies on TB at IREC are also supported by Grupo Santander—Fundación Marcelino Botin.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