Inhibition of the endosymbiont “Candidatus Midichloria mitochondrii” during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia

Background: The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks. Methods: Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM.Results: Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai. Conclusions: Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks

Plasma osteopontin concentrations in preeclampsia - is there an association with endothelial injury?

Background: It has been previously reported that plasma osteopontin (OPN) concentrations are increased in cardiovascular disorders. The goal of the present study was to determine plasma OPN concentrations in healthy pregnant women and preeclamptic patients, and to investigate their relationship to the clinical characteristics of the study subjects and to markers of inflammation [C-reactive protein (CRP)], endothelial activation [von Willebrand factor antigen (VWF: Ag)] or endothelial injury (fibronectin), oxidative stress [malondialdehyde (MDA)] and trophoblast debris (cell-free fetal DNA). Methods: Forty-four patients with preeclampsia and 44 healthy pregnant women matched for age and gestational age were involved in this case-control study. Plasma OPN concentrations were measured with ELISA. Serum CRP concentrations were determined with an autoanalyzer using the manufacturer's reagents. Plasma VWF: Ag was quantified by ELISA, while plasma fibronectin concentrations were measured by nephelometry. Plasma MDA concentrations were estimated by the thiobarbituric acid-based colorimetric assay. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time PCR analysis of the sex-determining region Y (SRY) gene. For statistical analyses, non-parametric methods were applied. Results: Serum levels of CRP, as well as plasma concentrations of VWF: Ag, fibronectin, MDA and cell-free fetal DNA were significantly higher in preeclamptic patients than in healthy pregnant women. There was no significant difference in plasma OPN concentrations between controls and the preeclamptic group. However, preeclamptic patients with plasma fibronectin concentrations in the upper quartile had significantly higher plasma OPN concentrations than those below the 75th percentile, as well as healthy pregnant women [median (interquartile range): 9.38 (8.10-11.99) vs. 7.54 (6.31-9.40) and 7.40 (6.51-8.80) ng/mL, respectively, p < 0.05 for both]. Furthermore, in preeclamptic patients, plasma OPN concentrations showed a significant positive linear association with plasma fibronectin (Spearman R = 0.38, standardized regression coefficient (beta) = 0.41, p < 0.05 for both). Conclusions: Plasma OPN concentrations are increased in preeclamptic patients with extensive endothelial injury. However, further studies are warranted to explore the relationship between OPN and endothelial damage. Clin Chem Lab Med 2010;48: 181-7

Acute febrile illness is associated with Rickettsia spp infection in dogs

BACKGROUND: Rickettsia conorii is transmitted by Rhipicephalus sanguineus ticks and causes Mediterranean Spotted Fever (MSF) in humans. Although dogs are considered the natural host of the vector, the clinical and epidemiological significance of R. conorii infection in dogs remains unclear. The aim of this prospective study was to investigate whether Rickettsia infection causes febrile illness in dogs living in areas endemic for human MSF. METHODS: Dogs from southern Italy with acute fever (n = 99) were compared with case–control dogs with normal body temperatures (n = 72). Serology and real-time PCR were performed for Rickettsia spp., Ehrlichia canis, Anaplasma phagocytophilum/A. platys and Leishmania infantum. Conventional PCR was performed for Babesia spp. and Hepatozoon spp. Acute and convalescent antibodies to R. conorii, E. canis and A. phagocytophilum were determined. RESULTS: The seroprevalence rates at first visit for R. conorii, E. canis, A. phagocytophilum and L. infantum were 44.8%, 48.5%, 37.8% and 17.6%, respectively. The seroconversion rates for R. conorii, E. canis and A. phagocytophilum were 20.7%, 14.3% and 8.8%, respectively. The molecular positive rates at first visit for Rickettsia spp., E. canis, A. phagocytophilum, A. platys, L. infantum, Babesia spp. and Hepatozoon spp. were 1.8%, 4.1%, 0%, 2.3%, 11.1%, 2.3% and 0.6%, respectively. Positive PCR for E. canis (7%), Rickettsia spp. (3%), Babesia spp. (4.0%) and Hepatozoon spp. (1.0%) were found only in febrile dogs. The DNA sequences obtained from Rickettsia and Babesia PCRs positive samples were 100% identical to the R. conorii and Babesia vogeli sequences in GenBank®, respectively. Febrile illness was statistically associated with acute and convalescent positive R. conorii antibodies, seroconversion to R. conorii, E. canis positive PCR, and positivity to any tick pathogen PCRs. Fourteen febrile dogs (31.8%) were diagnosed with Rickettsia spp. infection based on seroconversion and/or PCR while only six afebrile dogs (12.5%) seroconverted (P = 0.0248). The most common clinical findings of dogs with Rickettsia infection diagnosed by seroconversion and/or PCR were fever, myalgia, lameness, elevation of C-reactive protein, thrombocytopenia and hypoalbuminemia. CONCLUSIONS: This study demonstrates acute febrile illness associated with Rickettsia infection in dogs living in endemic areas of human MSF based on seroconversion alone or in combination with PCR

Protein-Glutamine Gamma-Glutamyltransferase

info:eu-repo/semantics/publishedVersio

Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: Inherent potential for biosynthetic versus live recombinant microbicides

<p>Abstract</p> <p>Background</p> <p>Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of ~120–200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2.</p> <p>Methods and Results</p> <p>Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but < 400 sites; 69(23.9%) in > 400 but < 700 sites; and the 9(3.1%) enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a nanoparticle for microbicide development and a "recombinant lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII.</p> <p>Conclusion</p> <p>Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.</p

Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy

<p>Abstract</p> <p>Background</p> <p><it>Ixodes ricinus</it>, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed.</p> <p>Methods</p> <p>From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, <it>Anaplasma phagocytophilum, Rickettsia </it>spp., <it>Babesia </it>spp., "<it>Candidatus Neoehrlichia mikurensis</it>"). Based on the results of this survey, three sampling strategies were evaluated <it>a</it>-<it>posteriori</it>, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C).</p> <p>Results</p> <p>Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were <it>Borrelia burgdorferi </it>sensu lato (17.6%), <it>Rickettsia helvetica </it>(13.1%), and "<it>Ca. N. mikurensis</it>" (10.5%). Within the <it>B. burgdorferi </it>complex, four genotypes (i.e., <it>B. valaisiana, B. garinii, B. afzelii</it>, and <it>B. burgdorferi </it>sensu stricto) were found. Less prevalent pathogens included <it>R. monacensis </it>(3.7%), TBE virus (2.1%), <it>A. phagocytophilum </it>(1.5%), <it>Bartonella </it>spp. (1%), and <it>Babesia </it>EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results.</p> <p>Conclusions</p> <p>Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.</p

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset