The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system

Background: Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR. Results: A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%). Conclusions: Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach

Factors Affecting Trypanosome Maturation in Tsetse Flies

Trypanosoma brucei brucei infections which establish successfully in the tsetse fly midgut may subsequently mature into mammalian infective trypanosomes in the salivary glands. This maturation is not automatic and the control of these events is complex. Utilising direct in vivo feeding experiments, we report maturation of T. b. brucei infections in tsetse is regulated by antioxidants as well as environmental stimuli. Dissection of the maturation process provides opportunities to develop transmission blocking vaccines for trypanosomiasis. The present work suggests L-cysteine and/or nitric oxide are necessary for the differentiation of trypanosome midgut infections in tsetse

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)−m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)−m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)−m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)−m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were \u3e0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance

Using molecular data for epidemiological inference: assessing the prevalence of Trypanosoma brucei rhodesiense in Tsetse in Serengeti, Tanzania

Background: Measuring the prevalence of transmissible Trypanosoma brucei rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessing human disease risk and monitoring spatio-temporal trends and the impact of control interventions. Although an important epidemiological variable, identifying flies which carry transmissible infections is difficult, with challenges including low prevalence, presence of other trypanosome species in the same fly, and concurrent detection of immature non-transmissible infections. Diagnostic tests to measure the prevalence of T. b. rhodesiense in tsetse are applied and interpreted inconsistently, and discrepancies between studies suggest this value is not consistently estimated even to within an order of magnitude. Methodology/Principal Findings: Three approaches were used to estimate the prevalence of transmissible Trypanosoma brucei s.l. and T. b. rhodesiense in Glossina swynnertoni and G. pallidipes in Serengeti National Park, Tanzania: (i) dissection/microscopy; (ii) PCR on infected tsetse midguts; and (iii) inference from a mathematical model. Using dissection/microscopy the prevalence of transmissible T. brucei s.l. was 0% (95% CI 0–0.085) for G. swynnertoni and 0% (0–0.18) G. pallidipes; using PCR the prevalence of transmissible T. b. rhodesiense was 0.010% (0–0.054) and 0.0089% (0–0.059) respectively, and by model inference 0.0064% and 0.00085% respectively. Conclusions/Significance: The zero prevalence result by dissection/microscopy (likely really greater than zero given the results of other approaches) is not unusual by this technique, often ascribed to poor sensitivity. The application of additional techniques confirmed the very low prevalence of T. brucei suggesting the zero prevalence result was attributable to insufficient sample size (despite examination of 6000 tsetse). Given the prohibitively high sample sizes required to obtain meaningful results by dissection/microscopy, PCR-based approaches offer the current best option for assessing trypanosome prevalence in tsetse but inconsistencies in relating PCR results to transmissibility highlight the need for a consensus approach to generate meaningful and comparable data

The Influence of Sex and Fly Species on the Development of Trypanosomes in Tsetse Flies

Unlike other dipteran disease vectors, tsetse flies of both sexes feed on blood and transmit pathogenic African trypanosomes. During transmission, Trypanosoma brucei undergoes a complex cycle of proliferation and development inside the tsetse vector, culminating in production of infective forms in the saliva. The insect manifests robust immune defences throughout the alimentary tract, which eliminate many trypanosome infections. Previous work has shown that fly sex influences susceptibility to trypanosome infection as males show higher rates of salivary gland (SG) infection with T. brucei than females. To investigate sex-linked differences in the progression of infection, we compared midgut (MG), proventriculus, foregut and SG infections in male and female Glossina morsitans morsitans. Initially, infections developed in the same way in both sexes: no difference was observed in numbers of MG or proventriculus infections, or in the number and type of developmental forms produced. Female flies tended to produce foregut migratory forms later than males, but this had no detectable impact on the number of SG infections. The sex difference was not apparent until the final stage of SG invasion and colonisation, showing that the SG environment differs between male and female flies. Comparison of G. m. morsitans with G. pallidipes showed a similar, though less pronounced, sex difference in susceptibility, but additionally revealed very different levels of trypanosome resistance in the MG and SG. While G. pallidipes was more refractory to MG infection, a very high proportion of MG infections led to SG infection in both sexes. It appears that the two fly species use different strategies to block trypanosome infection: G. pallidipes heavily defends against initial establishment in the MG, while G. m. morsitans has additional measures to prevent trypanosomes colonising the SG, particularly in female flies. We conclude that the tsetse-trypanosome interface works differently in G. m. morsitans and G. pallidipes

Perspective from a Younger Generation -- The Astro-Spectroscopy of Gisbert Winnewisser

Gisbert Winnewisser's astronomical career was practically coextensive with the whole development of molecular radio astronomy. Here I would like to pick out a few of his many contributions, which I, personally, find particularly interesting and put them in the context of newer results.Comment: 14 pages. (Co)authored by members of the MPIfR (Sub)millimeter Astronomy Group. To appear in the Proceedings of the 4th Cologne-Bonn-Zermatt-Symposium "The Dense Interstellar Medium in Galaxies" eds. S. Pfalzner, C. Kramer, C. Straubmeier, & A. Heithausen (Springer: Berlin

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut

Infections with Immunogenic Trypanosomes Reduce Tsetse Reproductive Fitness: Potential Impact of Different Parasite Strains on Vector Population Structure

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6–8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite–host coevolution, and disease prevalence are discussed

Inheriting library cards to Babel and Alexandria: Contemporary metaphors for the digital library

Librarians have been consciously adopting metaphors to describe library concepts since the nineteenth century, helping us to structure our understanding of new technologies. We have drawn extensively on these figurative frameworks to explore issues surrounding the digital library, yet very little has been written to date which interrogates how these metaphors have developed over the years. Previous studies have explored library metaphors, using either textual analysis or ethnographic methods to investigate their usage. However, this is to our knowledge the first study to use bibliographic data, corpus analysis, qualitative sentiment weighting and close reading to study particular metaphors in detail. It draws on a corpus of over 450 articles to study the use of the metaphors of the Library of Alexandria and Babel, concluding that both have been extremely useful as framing metaphors for the digital library. However, their longstanding use has seen them become stretched as metaphors, meaning that the field’s figurative framework now fails to represent the changing technologies which underpin contemporary digital libraries