Epidemiology and molecular characterisation of Ehrlichia phagocytophila in relation to emerging ehrlichiae

Ehrlichia phagocytophila (Genus Ehrlichia, Order Rickettsiales) is the pathogen responsible for Tick-borne fever, a disease of high morbidity in susceptible ruminants. These bacteria appear to be almost identical at serological and molecular level to granulocytic Ehrlichia species recently diagnosed in humans, dogs and horses of Europe and the United States.A molecular description of different isolates of the pathogen is given. Samples were derived from wild and domestic vertebrate hosts from the UK, where Tick-borne fever is endemic. Molecular characterisation of a fragment from the groE operon gene showed higher nucleotide variation than at 16S rDNA level. Human and equine isolates from Europe differed from North American samples, which at 16S appeared to be identical. Further differences were also found between ruminant and non-ruminant granulocytic samples from Europe. Genomic analysis of less conserved genes appears necessary to provide more useful phylogenetic information that will help to clarify the relationship between closely related bacterial species.Populations of the vector tick, Ixodes ricinus, were sampled and analysed to determine the prevalence of infection and clarify their role in the epidemiology of the disease. The studies indicated a low infection prevalence that seems, however, enough to maintain the pathogen in nature. The prevalence varied according to widespread sites across Britain but it was always lower than expected from information in the literature. Attempts to determine the efficiency of latent infection in sheep to transmit Ehrlichia to ticks were unsuccessful.A seroepidemiological survey was undertaken using IFAT and involving samples from suspected vertebrate reservoirs of infection such as dogs, cats, horses, and deer in order to determine if those species were exposed to the pathogen and the range of hosts for the bacteria in widespread sites across Britain. The results suggested high rates of exposure in dogs from rural areas and wild roe deer. Cats showed also a high seroprevalence indicating the three vertebrate hosts were exposed to E. phagocytophila and mounted an immune response towards the pathogen. It remains to be elucidated if dogs, cats and horses are accidental or competent reservoirs of infection. The presence of E. phagocytophila in roe deer blood and spleen samples was confirmed by PCR. Tick counts from deer legs ratified that all three stages of tick (larvae, nymphs and adults) were able to feed simultaneously upon roe deer thus supporting their role as competent reservoirs for both ticks and E. phagocytophila together with the serological and molecular evidence.Cytoecetes ondiri, an African relative of Ehrlichia phagocytophila, was shown to cross-react in immunoblots with E. equi and in IFAT with E. phagocytophila antigens thus confirming a close antigenic relationship.ELISA were developed using crude E. equi and E. phagocytophila as antigens and samples from several vertebrate species. The assays were validated with previous results obtained by IFAT. Data suggested that E. equi is a useful surrogate antigen for serologic studies until E. phagocytophila is routinely grown in culture. The antigenic structure of Ehrlichia was further characterised using mitochondria as surrogate antigens under the evidence of the phylogenetic relationship between the organelles and the bacteria. Ehrlichia are classified in the a-subgroup of Proteobacteria, which are believed to be the closest relatives to mitochondria. Sera from experimentally inoculated animals recognised mitochondrial antigens prior and after exposure but the responses were significantly higher after infection and challenge. Further work should be directed towards the successful cultivation of the pathogen as for HGE and E. equi in order to develop more reliable serological tests for E. phagocytophila for future epidemiological surveys. Identification of the major antigenic components of the organism will also help towards vaccine development

Common Strategies, Different Mechanisms to Infect the Host: Anaplasma and Mycobacterium

Intracellular bacteria such as Anaplasma spp. and Mycobacterium spp. pose a risk to human and animal populations worldwide. The main function of immune response cells is to eliminate invading pathogens. However, pathogens can deregulate host cell function and turn defense cells into suitable hosts. Intracellular bacterial have a smaller genome, compared to the host cell, thus requiring efficient mechanisms for survival and persistence within the host by inducing sustained changes in cell function and immune response. Bacterial epigenetic regulation of host cell gene transcription appears to be a general mechanism that enhances pathogen survival while altering host cell function and facilitating infection. Anaplasma phagocytophilum leads to modified host cell gene transcription and phenotype by epigenetically altering host chromatin. Mycobacterial infection of human cells also results in host gene silencing using a mechanism that involves HDAC complex formation and histone deacetylation. Membrane proteins are essential for cell invasion in both pathogens, and can regulate and protect the pathogen against the host response. Understanding the mechanisms employed by these bacteria to infect the host could contribute to develop effective interventions for the control of tuberculosis and anaplasmosis. This review focuses on the common strategies employed by two zoonotic pathogens, Anaplasma and Mycobacterium spp., highlighting also the different mechanisms used to infect host cells

Vacunas en situaciones especiales. Embarazo, inmunodepresión, transplante

En las recomendaciones de vacunación de grupos de población que por su situación de salud requieren medidas específicas deben conjugarse: Las limitaciones en la aplicación de determinadas vacunas por la mayor probabilidad de efectos secundarios en estos pacientes (por ejemplo la aplicación de vacunas de virus vivos en pacientes inmunodeprimidos). El mayor riesgo de infección frente a determinados agentes que genera la patología o situación inmunitaria del paciente, y que determina un mayor interés sanitario de la aplicación de las vacunas frente a los mismos. La menor respuesta inmunitaria a las vacunas, por la menor capacidad de respuesta del sistema inmune de estos pacientes. La consideración de estos tres factores, en el marco de una creciente evidencia de la importancia sanitaria de las estrategias de vacunación diseñadas específicamente para cada grupo de pacientes por los efectos protectores y la disminución de la incidencia o gravedad de las infecciones frente a las que se ha inmunizado, está llevando, en la última década, al diseño de calendarios de vacunación específicos, así como a un creciente impulso de estrategias de vacunación específicas para garantizar la captación y cumplimiento de estos calendarios. En este artículo se revisan las siguientes situaciones: Embarazo; Inmunosupresión ; Inmunodepresión ; Trasplante de progenitores hematopoyéticos ; Asplenia ; Trasplante de órganos sólidos. Se reseña la importancia de que toda mujer esté protegida frente a la rubéola antes del embarazo, y de la administración de esta vacuna en el post-parto a aquellas mujeres que no hubieran sido vacunadas previamente. Así mismo se insiste en la necesidad de la vacunación antigripal anual en el 2º o 3º trimestre del embarazo y de la importancia de la vacunación frente al tétanos antes del parto en las mujeres que no hayan completado previamente su vacunación. El paciente receptor de progenitores hematopoyéticos, independientemente de la edad, requiere la revacunación con todas las vacunas del calendario infantil, a las que debe añadirse la vacuna antineumocócica, antihaemophilus influenzae tipo b y antigripal, además de otras vacunas vinculadas a riesgos individuales. Los pacientes asplénicos presentan un alto riesgo de infecciones provocadas por bacterias capsuladas, por lo que cobra especial trascendencia la vacunación frente al neumococo, el Hib y el meningococo, además de la vacunación antigripal anual. Los pacientes sometidos a programa de trasplante de órgano sólido suponen un colectivo específico en el que debe programarse la aplicación de las vacunas antes del trasplante dada la mejor respuesta inmune en ese momento, y la vacunación postrasplante, según la situación inmune previa. Se destaca la importancia de la prevención pretrasplante frente a la varicela y la hepatitis B, así como las recomendaciones específicas de vacunación según el tipo de trasplante: frente al neumococo, el Haemophilus influenzae tipo b, la gripe, la Hepatitis A, etc, además de completar el calendario de vacunación infantil en los menores de 14 años, y de estar vacunado frente al tétanos, difteria, sarampión, rubéola, parotiditis y gripe anualmente en los adultos. Un aspecto a resaltar es la importancia de la vacunación del entorno del paciente inmunodeprimido, en especial en caso de trasplante, así como las consideraciones a tener en cuenta en las vacunas recomendadas a los convivientes

Comparison of micrometeorological techinques for estimating ammonia emission from covered slurry storage and land spreading of cattle slurry

The micrometeorological mass-balance integrated horizontal flux (IHF) technique has been commonly employed for measuring ammonia (NH3) emissions inon-field experiments. However, the inverse-dispersion modeling technique, such as the backward Lagrangian stochastic (bLS) modeling approach, is currently highlighted as offering flexibility in plot design and requiring a minimum number of samplers (Ro et al., 2013). The objective of this study was to make a comparison between the bLS technique with the IHF technique for estimating NH3 emission from flexible bag storage and following landspreading of dairy cattle slurry. Moreover, considering that NH3 emission in storage could have been non uniform, the effect on bLS estimates of a single point and multiple downwind concentration measurements was tested, as proposed by Sanz et al. (2010)

Ammonia and greenhouse gas emissions from an enriched cage laying hen facility

[EN] Ammonia, methane, nitrous oxide and carbon dioxide emissions were measured during a complete production cycle in an enriched cage laying hen facility under Oceanic climate conditions. Continuous monitoring of gas concentration, ventilation rate and environmental parameters were conducted from April 2012 to September 2013. The seasonal and diurnal pattern of gas emissions was analysed. Seasonality effect was found for NH3 emission, showing an average emission of 144.9 mg d 1 hen 1 and 90.3 mg d 1 hen 1 in summer and winter, respectively. On the contrary, diurnal pattern of NH3 emission did not differ between these seasons. For CO2, mean emission values did not show seasonality, although the diurnal pattern differed between winter and summer. Results obtained for CH4 and N2O emissions did not provide sufficient evidence to determine either seasonality or diurnal effect on these gases. An NH3 emission factor of 7% of total N in manure was defined for this system. These losses increased at higher ventilation rates and lower belt cleaning frequencies. Thus, NH3 mitigation strategies at housing level should consider both parameters. Further studies would be necessary to determine how these factors regulate NH3 emission at laying hen houses. © 2016 IAgrE. Published by Elsevier Ltd. All rights reservedThis work has been funded by BATFARM Interreg-Atlantic Area Project (2009-1/071) entitled "Evaluation of best available techniques to decrease air and water pollution in animal farms". Oier Alberdi holds a grant from the Ph.D. student's research program of the Department of Economic Development and Competitiveness of the Basque Government. The authors are especially grateful to Larrabe Oilotegia S.A.T. that facilitated productive data and access to the farm and to the engineering company Ingenieria Avicola S.L. for the detailed information on ventilation aspects of the installation.Alberdi, O.; Arriaga, H.; Calvet, S.; Estellés, F.; Merino, P. (2016). Ammonia and greenhouse gas emissions from an enriched cage laying hen facility. Biosystems Engineering. 144:1-12. doi:10.1016/j.biosystemseng.2016.01.009S11214

Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

Interferon (IFN)-alpha is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-alpha subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-alpha2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-alpha subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-alpha mRNAs and in the production of IFN-alpha protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-alpha5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-alpha8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-alpha subtypes, IFN-alpha5 being the specific subtype induced in PBMC from patients with chronic HCV infection

The intracellular bacterium Anaplasma phagocytophilum selectively manipulates the levels of vertebrate host proteins in the tick vector Ixodes scapularis

Background: The intracellular bacteria Anaplasma phagocytophilum are emerging zoonotic pathogens affecting human and animal health, and a good model for the study of tick-host-pathogen interactions. This tick-borne pathogen is transmitted by Ixodes scapularis in the United States where it causes human granulocytic anaplasmosis. Tick midguts and salivary glands play a major role during tick feeding and development, and in pathogen acquisition, multiplication and transmission. Vertebrate host proteins are found in tick midguts after feeding and have been described in the salivary glands of fed and unfed ticks, suggesting a role for these proteins during tick feeding and development. Furthermore, recent results suggested the hypothesis that pathogen infection affects tick metabolic processes to modify host protein digestion and persistence in the tick with possible implications for tick physiology and pathogen life-cycle. Methods: To address this hypothesis, herein we used I. scapularis female ticks fed on uninfected and A. phagocytophilum-infected sheep to characterize host protein content in midguts and salivary glands by proteomic analysis of tick tissues. Results: The results evidenced a clear difference in the host protein content between tick midguts and salivary glands in response to infection suggesting that A. phagocytophilum selectively manipulates the levels of vertebrate host proteins in ticks in a tissue-specific manner to facilitate pathogen infection, multiplication and transmission while preserving tick feeding and development. The mechanisms by which A. phagocytophilum manipulates the levels of vertebrate host proteins are not known, but the results obtained here suggested that it might include the modification of proteolytic pathways. Conclusions: The results of this study provided evidence to support that A. phagocytophilum affect tick proteolytic pathways to selectively manipulate the levels of vertebrate host proteins in a tissue-specific manner to increase tick vector capacity. Investigating the biological relevance of host proteins in tick biology and pathogen infection and the mechanisms used by A. phagocytophilum to manipulate host protein content is essential to advance our knowledge of tick-host-pathogen molecular interactions. These results have implications for the identification of new targets for the development of vaccines for the control of tick-borne diseases.This research was supported by the Ministerio de Economia y Competitividad (Spain) grant BFU2011-23896 and the European Union (EU) Seventh Framework Programme (FP7) ANTIGONE project number 278976. NA was funded by Ministerio de Economia y Competitividad, Spain. MV was supported by the Research Plan of the University of Castilla - La Mancha, Spain. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

Draft Genome Sequences of Anaplasma phagocytophilum, A. marginale, and A. ovis Isolates from Different Hosts

Here, we report the draft genome sequences of isolates of Anaplasma phagocytophilum, Anaplasma marginale, and Anaplasma ovis. The genomes of A. phagocytophilum (human), A. marginale (cattle), and A. ovis (goat) isolates from the United States were sequenced and characterized. This is the first report of an A. ovis genome sequence

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

Tick galactosyltransferases are involved in a-Gal synthesis and play a role during Anaplasma phagocytophilum infection and Ixodes scapularis tick vector development

The carbohydrate Gala1-3Galß1-(3)4GlcNAc-R (a-Gal) is produced in all mammals except for humans, apes and old world monkeys that lost the ability to synthetize this carbohydrate. Therefore, humans can produce high antibody titers against a-Gal. Anti-a-Gal IgE antibodies have been associated with tick-induced allergy (i.e. a-Gal syndrome) and anti-a-Gal IgG/IgM antibodies may be involved in protection against malaria, leishmaniasis and Chagas disease. The a-Gal on tick salivary proteins plays an important role in the etiology of the a-Gal syndrome. However, whether ticks are able to produce endogenous a-Gal remains currently unknown. In this study, the Ixodes scapularis genome was searched for galactosyltransferases and three genes were identified as potentially involved in the synthesis of a-Gal. Heterologous gene expression in a-Gal-negative cells and gene knockdown in ticks confirmed that these genes were involved in a-Gal synthesis and are essential for tick feeding. Furthermore, these genes were shown to play an important role in tick-pathogen interactions. Results suggested that tick cells increased a-Gal levels in response to Anaplasma phagocytophilum infection to control bacterial infection. These results provided the molecular basis of endogenous a-Gal production in ticks and suggested that tick galactosyltransferases are involved in vector development, tick-pathogen interactions and possibly the etiology of a-Gal syndrome in humans