West Africa - A Safe Haven for Frogs? A Sub-Continental Assessment of the Chytrid Fungus (Batrachochytrium dendrobatidis)

A putative driver of global amphibian decline is the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd). While Bd has been documented across continental Africa, its distribution in West Africa remains ambiguous. We tested 793 West African amphibians (one caecilian and 61 anuran species) for the presence of Bd. The samples originated from seven West African countries - Bénin, Burkina Faso, Côte d''Ivoire, Ghana, Guinea, Liberia, Sierra Leone - and were collected from a variety of habitats, ranging from lowland rainforests to montane forests, montane grasslands to humid and dry lowland savannahs. The species investigated comprised various life-history strategies, but we focused particularly on aquatic and riparian species. We used diagnostic PCR to screen 656 specimen swabs and histology to analyse 137 specimen toe tips. All samples tested negative for Bd, including a widespread habitat generalist Hoplobatrachus occipitalis which is intensively traded on the West African food market and thus could be a potential dispersal agent for Bd. Continental fine-grained (30 arc seconds) environmental niche models suggest that Bd should have a broad distribution across West Africa that includes most of the regions and habitats that we surveyed. The surprising apparent absence of Bd in West Africa indicates that the Dahomey Gap may have acted as a natural barrier. Herein we highlight the importance of this Bd-free region of the African continent - especially for the long-term conservation of several threatened species depending on fast flowing forest streams (Conraua alleni (“Vulnerable”) and Petropedetes natator (“Near Threatened”)) as well as the “Critically Endangered” viviparous toad endemic to the montane grasslands of Mount Nimba (Nimbaphrynoides occidentalis).     

Opt-Out Panel Testing for HIV,Hepatitis B and Hepatitis C in an Urban Emergency Department: A Pilot Study

Objectives

Studies suggest 2 per 1000 people in Dublin are living with HIV, the level above which universal screening is advised. We aimed to assess the feasibility and acceptability of a universal opt-out HIV, Hepatitis B and Hepatitis C testing programme for Emergency Department patients and to describe the incidence and prevalence of blood-borne viruses in this population.

Methods

An opt-out ED blood borne virus screening programme was piloted from March 2014 to January 2015. Patients undergoing blood sampling during routine clinical care were offered HIV 1&2 antibody/antigen assay, HBV surface antigen and HCV antibody tests. Linkage to care where necessary was co-ordinated by the study team. New diagnosis and prevalence rates were defined as the new cases per 1000 tested and number of positive tests per 1000 tested respectively.

Results

Over 45 weeks of testing, of 10,000 patient visits, 8,839 individual patient samples were available for analysis following removal of duplicates. A sustained target uptake of >50% was obtained after week 3. 97(1.09%), 44(0.49%) and 447(5.05%) HIV, Hepatitis B and Hepatitis C tests were positive respectively. Of these, 7(0.08%), 20(0.22%) and 58(0.66%) were new diagnoses of HIV, Hepatitis B and Hepatitis C respectively. The new diagnosis rate for HIV, Hepatitis B and Hepatitis C was 0.8, 2.26 and 6.5 per 1000 and study prevalence for HIV, Hepatitis B and Hepatitis C was 11.0, 5.0 and 50.5 per 1000 respectively.

Conclusions

Opt-out blood borne viral screening was feasible and acceptable in an inner-city ED. Blood borne viral infections were prevalent in this population and newly diagnosed cases were diagnosed and linked to care. These results suggest widespread blood borne viral testing in differing clinical locations with differing population demographic risks may be warranted.     

M-Cells Contribute to the Entry of an Oral Vaccine but Are Not Essential for the Subsequent Induction of Protective Immunity against Francisella tularensis

M-cells (microfold cells) are thought to be a primary conduit of intestinal antigen trafficking. Using an established neutralizing anti-RANKL (Receptor Activator of NF-κB Ligand) antibody treatment to transiently deplete M-cells in vivo, we sought to determine whether intestinal M-cells were required for the effective induction of protective immunity following oral vaccination with ΔiglB (a defined live attenuated Francisella novicida mutant). M-cell depleted, ΔiglB-vaccinated mice exhibited increased (but not significant) morbidity and mortality following a subsequent homotypic or heterotypic pulmonary F. tularensis challenge. No significant differences in splenic IFN-γ, IL-2, or IL-17 or serum antibody (IgG1, IgG2a, IgA) production were observed compared to non-depleted, ΔiglB-vaccinated animals suggesting complementary mechanisms for ΔiglB entry. Thus, we examined other possible routes of gastrointestinal antigen sampling following oral vaccination and found that ΔiglB co-localized to villus goblet cells and enterocytes. These results provide insight into the role of M-cells and complementary pathways in intestinal antigen trafficking that may be involved in the generation of optimal immunity following oral vaccination.     

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring

Background

As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys.

Methods and Findings

The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting.

Conclusion

We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.     

Finding stories in noise: Mitochondrial portraits from RAD data

Mitochondrial DNA (mtDNA) has formed the backbone of phylogeographic research for many years; however, recent trends focus on genome‐wide analyses. One method proposed for calibrating inferences from noisy next‐generation data, such as RAD sequencing, is to compare these results with analyses of mitochondrial sequences. Most researchers using this approach appear to be unaware that many single nucleotide polymorphisms (SNPs) identified from genome‐wide sequence data are themselves mitochondrial, or assume that these are too few to bias analyses. Here, we demonstrate two methods for mining mitochondrial markers using RAD sequence data from three South African species of yellowfish, Labeobarbus. First, we use a rigorous SNP discovery pipeline using the program stacks , to identify variant sites in mtDNA, which we then combine into haplotypes. Second, we directly map sequence reads against a mitochondrial genome reference. This method allowed us to reconstruct up to 98% of the Labeobarbus mitogenome. We validated these mitogenome reconstructions through blast database searches and by comparison with cytochrome b gene sequences obtained through Sanger sequencing. Finally, we investigate the organismal consequences of these data including ancient genetic exchange and a recent translocation among populations of L. natalensis, as well as interspecific hybridization between L. aeneus and L. kimberleyensis.     

Substrate specificity of RdgB protein, a deoxyribonucleoside triphosphate pyrophosphohydrolase

We have previously reported the identification of a DNA repair system in Escherichia coli for the prevention of the stable incorporation of noncanonical purine dNTPs into DNA. We hypothesized that the RdgB protein is active on 2'-deoxy-N-6-hydroxylaminopurine triphosphate (dHAPTP) as well as deoxyinosine triphosphate. Here we show that RdgB protein and RdgB homologs from Saccharomyces cerevisiae, mouse, and human all possess deoxyribonucleoside triphosphate pyrophosphohydrolase activity and that all four RdgB homologs have high specificity for dHAPTP and deoxyinosine triphosphate compared with the four canonical dNTPs and several other noncanonical (d)NTPs. Kinetic analysis reveals that the major source of the substrate specificity lies in changes in K(m) for the various substrates. The expression of these enzymes in E. coli complements defects that are caused by the incorporation of HAP and an endogenous noncanonical purine into DNA. Our data support a preemptive role for the RdgB homologs in excluding endogenous and exogenous modified purine dNTPs from incorporation into DNA.     

Conservation of regional gene expression in mouse and human brain

Many neurodegenerative diseases have a hallmark regional and cellular pathology. Gene expression analysis of healthy tissues may provide clues to the differences that distinguish resistant and sensitive tissues and cell types. Comparative analysis of gene expression in healthy mouse and human brain provides a framework to explore the ability of mice to model diseases of the human brain. It may also aid in understanding brain evolution and the basis for higher order cognitive abilities. Here we compare gene expression profiles of human motor cortex, caudate nucleus, and cerebellum to one another and identify genes that are more highly expressed in one region relative to another. We separately perform identical analysis on corresponding brain regions from mice. Within each species, we find that the different brain regions have distinctly different expression profiles. Contrasting between the two species shows that regionally enriched genes in one species are generally regionally enriched genes in the other species. Thus, even when considering thousands of genes, the expression ratios in two regions from one species are significantly correlated with expression ratios in the other species. Finally, genes whose expression is higher in one area of the brain relative to the other areas, in other words genes with patterned expression, tend to have greater conservation of nucleotide sequence than more widely expressed genes. Together these observations suggest that region-specific genes have been conserved in the mammalian brain at both the sequence and gene expression levels. Given the general similarity between patterns of gene expression in healthy human and mouse brains, we believe it is reasonable to expect a high degree of concordance between microarray phenotypes of human neurodegenerative diseases and their mouse models. Finally, these data on very divergent species provide context for studies in more closely related species that address questions such as the origins of cognitive differences.     

A tolloid homologue from the Pacific oyster Crassostrea gigas

The genes governing mesoderm specification have been extensively studied in vertebrates, arthropods and nematodes. The latter two phyla belong to the Ecdysozoan clade but little is understood of the role that these genes might play in the development of the other major protostomal clade, the Lophotrochozoa. As part of a wider project to analyze the functions associated with transforming growth factor beta superfamily members in Lophotrochozoa, we have cloned a gene encoding a tolloid homologue from the bivalve mollusc Crassostrea gigas. Tolloid is a key developmental protein that regulates the activity of bone morphogenetic proteins (BMPs). We have determined the intron-exon structure of the gene encoding C. gigas tolloid and have compared it with those of homologous genes from both protostomes and deuterostomes. In order to analyze the functionality of oyster tolloid the zebrafish embryo has been employed as a reporter organism and we show that over-expression of this protein results in the ventralization of zebrafish embryos at 24h post fertilization. The expression of the C. gigas tolloid gene during embryonic and larval development as well as in adult tissues is also explored.     

Light capture and pigment diversity in marine and freshwater cryptophytes

Phenotypic traits associated with light capture and phylogenetic relationships were characterized in 34 strains of diversely pigmented marine and freshwater cryptophytes. Nuclear SSU and partial LSU rDNA sequence data from 33 of these strains plus an additional 66 strains produced a concatenated rooted maximum likelihood tree that classified the strains into 7 distinct clades. Molecular and phenotypic data together support: (i) the reclassification of Cryptomonas irregularis NIES 698 to the genus Rhodomonas, (ii) revision of phycobiliprotein (PBP) diversity within the genus Hemiselmis to include cryptophyte phycocyanin (Cr‐PC) 569, (iii) the inclusion of previously unidentified strain CCMP 2293 into the genus Falcomonas, even though it contains cryptophyte phycoerythrin 545 (Cr‐PE 545), and (iv) the inclusion of previously unidentified strain CCMP 3175, which contains Cr‐PE 545, in a clade with PC‐containing Chroomonas species. A discriminant analysis‐based model of group membership correctly predicted 70.6% of the clades using three traits: PBP concentration · cell^?1, the wavelength of PBP maximal absorption, and habitat. Non‐PBP pigments (alloxanthin, chl‐a, chl‐c₂, α‐carotene) did not contribute significantly to group classification, indicating the potential plasticity of these pigments and the evolutionary conservation of the PBPs. Pigment data showed evidence of trade‐offs in investments in PBPs vs. chlorophylls (a +c₂).