One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus,Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.     

Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes

The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α(1) PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α(1) domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquisition of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.     

Dry season reproductive depression of Anopheles gambiae in the Sahel

The African malaria mosquito, Anopheles gambiae, is widespread south of the Sahara including in dry savannahs and semi-arid environments where no surface water exists for several months a year. Adults of the M form of An. gambiae persist through the long dry season, when no surface waters are available, by increasing their maximal survival from 4 weeks to 7 months. Dry season diapause (aestivation) presumably underlies this extended survival. Diapause in adult insects is intrinsically linked to depressed reproduction. To determine if reproduction of the Sahelian M form is depressed during the dry season, we assessed seasonal changes in oviposition, egg batch size, and egg development, as well as insemination rate and blood feeding in wild caught mosquitoes. Results from xeric Sahelian and riparian populations were compared. Oviposition response in the Sahelian M form dropped from 70% during the wet season to 20% during the dry season while the mean egg batch size among those that laid eggs fell from 173 to 101. Correspondingly, the fraction of females that exhibited gonotrophic dissociation increased over the dry season from 5% to 45%, while a similar fraction of the population retained developed eggs despite having access to water. This depression in reproduction the Sahelian M form was not caused by a reduced insemination rate. Seasonal variation in these reproductive parameters of the riparian M form population was less extreme and the duration of reproductive depression was shorter. Blood feeding responses did not change with the season in either population. Depressed reproduction during the dry season in the Sahelian M form of An. gambiae provides additional evidence for aestivation and illuminates the physiological processes involved. The differences between the Sahelian and riparian population suggest an adaptive cline in aestivation phenotypes between populations only 130 km apart.     

Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro,Burkina Faso

The adoption of Artemisinin based combination therapies (ACT) constitutes a basic strategy for malaria control in sub-Saharan Africa. Moreover, since cases of ACT resistance have been reported in South-East Asia, the need to understand P. falciparum resistance mechanism to ACT has become a global research goal. The selective pressure of ACT and the possibility that some specific Pfcrt and Pfmdr1 alleles are associated with treatment failures was assessed in a clinical trial comparing ASAQ to AL in Nanoro. Dried blood spots collected on Day 0 and on the day of recurrent parasitaemia during the 28-day follow-up were analyzed using the restriction fragments length polymorphism (PCR-RFLP) method to detect single nucleotide polymorphisms (SNPs) in Pfcrt (codon76) and Pfmdr1 (codons 86, 184, 1034, 1042, and 1246) genes. Multivariate analysis of the relationship between the presence of Pfcrt and Pfmdr1 alleles and treatment outcome was performed. AL and ASAQ exerted opposite trends in selecting Pfcrt K76T and Pfmdr1-N86Y alleles, raising the potential beneficial effect of using diverse ACT at the same time as first line treatments to reduce the selective pressure by each treatment regimen. No clear association between the presence of Pfcrt and Pfmdr1 alleles carried at baseline and treatment failure was observed.     

Augmentative on-farm delivery methods for the parasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae) to control the millet head miner Heliocheilus albipunctella (de Joannis) (Lepidoptera: Noctuidae) in Burkina Faso and Niger

Augmentative on-farm delivery methods for the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) to control the millet head miner (MHM) Heliocheilus albipunctella (de Joannis) (Lepidoptera: Noctuidae) were investigated in Burkina Faso from 2011 to 2012 and in Niger in 2012. Our findings indicate that 7 cm × 10 cm jute bags containing 50 g of millet grains, 30 g of millet flour, 25 Corcyra cephalonica larvae and two mated H. hebetor females are the most effective option for on-farm delivery of the parasitoid. The parasitoid progeny started emerging from the bags eight days after confinement and 57–71 parasitoid adults emerged from each bag. Using the methods we developed, over 90 % parasitism of MHM larvae was achieved in millet farms. The implications of these findings for a large extension of MHM biocontrol program are discussed.     

Niger’s Child Survival Success,Contributing Factors and Challenges to Sustainability: A Retrospective Analysis

BackgroundHousehold surveys undertaken in Niger since 1998 have revealed steady declines in under-5 mortality which have placed the country ‘on track’ to reach the fourth Millennium Development goal (MDG). This paper explores Niger’s mortality and health coverage data for children under-5 years of age up to 2012 to describe trends in high impact interventions and the resulting impact on childhood deaths averted. The sustainability of these trends are also considered.ConclusionIncreases in access and coverage of care for mothers and children have averted a considerable number of childhood deaths. The 2006 free health care policy and health post expansion were paramount in reducing barriers to care. However the sustainability of this policy and health service provision is precarious in light of persistently high fertility rates, unpredictable GDP growth, a high dependence on donor support and increasing pressures on government funding.     

Cost‐effective enrichment hybridization capture of chloroplast genomes at deep multiplexing levels for population genetics and phylogeography studies

Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large data sets thanks to next‐generation sequencing methods. In this study, we develop an easy and cost‐effective protocol for in‐solution enrichment hybridization capture of complete chloroplast genomes applicable at deep‐multiplexed levels. The protocol uses cheap in‐house species‐specific probes developed via long‐range PCR of the entire chloroplast. Barcoded libraries are constructed, and in‐solution enrichment of the chloroplasts is carried out using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes.