Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform

Targeted Next Generation Sequencing for malaria research in Africa:Current status and outlook

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed

Using a New Odour-Baited Device to Explore Options for Luring and Killing Outdoor-Biting Malaria Vectors: A Report on Design and Field Evaluation of the Mosquito Landing Box.

Mosquitoes that bite people outdoors can sustain malaria transmission even where effective indoor interventions such as bednets or indoor residual spraying are already widely used. Outdoor tools may therefore complement current indoor measures and improve control. We developed and evaluated a prototype mosquito control device, the 'Mosquito Landing Box' (MLB), which is baited with human odours and treated with mosquitocidal agents. The findings are used to explore technical options and challenges relevant to luring and killing outdoor-biting malaria vectors in endemic settings. Field experiments were conducted in Tanzania to assess if wild host-seeking mosquitoes 1) visited the MLBs, 2) stayed long or left shortly after arrival at the device, 3) visited the devices at times when humans were also outdoors, and 4) could be killed by contaminants applied on the devices. Odours suctioned from volunteer-occupied tents were also evaluated as a potential low-cost bait, by comparing baited and unbaited MLBs. There were significantly more Anopheles arabiensis, An. funestus, Culex and Mansonia mosquitoes visiting baited MLB than unbaited controls (P<=0.028). Increasing sampling frequency from every 120 min to 60 and 30 min led to an increase in vector catches of up to 3.6 fold (P<=0.002), indicating that many mosquitoes visited the device but left shortly afterwards. Outdoor host-seeking activity of malaria vectors peaked between 7:30 and 10:30pm, and between 4:30 and 6:00am, matching durations when locals were also outdoors. Maximum mortality of mosquitoes visiting MLBs sprayed or painted with formulations of candidate mosquitocidal agent (pirimiphos-methyl) was 51%. Odours from volunteer occupied tents attracted significantly more mosquitoes to MLBs than controls (P<0.001). While odour-baited devices such as the MLBs clearly have potential against outdoor-biting mosquitoes in communities where LLINs are used, candidate contaminants must be those that are effective at ultra-low doses even after short contact periods, since important vector species such as An. arabiensis make only brief visits to such devices. Natural human odours suctioned from occupied dwellings could constitute affordable sources of attractants to supplement odour baits for the devices. The killing agents used should be environmentally safe, long lasting, and have different modes of action (other than pyrethroids as used on LLINs), to curb the risk of physiological insecticide resistance

What's normal? Oligosaccharide concentrations and profiles in milk produced by healthy women vary geographically.

Background: Human milk is a complex fluid comprised of myriad substances, with one of the most abundant substances being a group of complex carbohydrates referred to as human milk oligosaccharides (HMOs). There has been some evidence that HMO profiles differ in populations, but few studies have rigorously explored this variability.Objectives: We tested the hypothesis that HMO profiles differ in diverse populations of healthy women. Next, we examined relations between HMO and maternal anthropometric and reproductive indexes and indirectly examined whether differences were likely related to genetic or environmental variations.Design: In this cross-sectional, observational study, milk was collected from a total of 410 healthy, breastfeeding women in 11 international cohorts and analyzed for HMOs by using high-performance liquid chromatography.Results: There was an effect of the cohort (P 4 times higher in milk collected in Sweden than in milk collected in rural Gambia (mean ± SEM: 473 ± 55 compared with 103 ± 16 nmol/mL, respectively; P < 0.05), and disialyllacto-N-tetraose (DSLNT) concentrations ranged from 216 ± 14 nmol/mL (in Sweden) to 870 ± 68 nmol/mL (in rural Gambia) (P < 0.05). Maternal age, time postpartum, weight, and body mass index were all correlated with several HMOs, and multiple differences in HMOs [e.g., lacto-N-neotetrose and DSLNT] were shown between ethnically similar (and likely genetically similar) populations who were living in different locations, which suggests that the environment may play a role in regulating the synthesis of HMOs.Conclusions: The results of this study support our hypothesis that normal HMO concentrations and profiles vary geographically, even in healthy women. Targeted genomic analyses are required to determine whether these differences are due at least in part to genetic variation. A careful examination of sociocultural, behavioral, and environmental factors is needed to determine their roles in this regard. This study was registered at clinicaltrials.gov as NCT02670278

Host choice and multiple blood feeding behaviour of malaria vectors and other anophelines in Mwea rice scheme, Kenya

<p>Abstract</p> <p>Background</p> <p>Studies were conducted between April 2004 and February 2006 to determine the blood-feeding pattern of <it>Anopheles </it>mosquitoes in Mwea Kenya.</p> <p>Methods</p> <p>Samples were collected indoors by pyrethrum spay catch and outdoors by Centers for Disease Control light traps and processed for blood meal analysis by an Enzyme-linked Immunosorbent Assay.</p> <p>Results</p> <p>A total of 3,333 blood-fed <it>Anopheles </it>mosquitoes representing four <it>Anopheles </it>species were collected and 2,796 of the samples were assayed, with <it>Anopheles arabiensis </it>comprising 76.2% (n = 2,542) followed in decreasing order by <it>Anopheles coustani </it>8.9% (n = 297), <it>Anopheles pharoensis </it>8.2% (n = 272) and <it>Anopheles funestus </it>6.7% (n = 222). All mosquito species had a high preference for bovine (range 56.3–71.4%) over human (range 1.1–23.9%) or goat (0.1–2.2%) blood meals. Some individuals from all the four species were found to contain mixed blood meals. The bovine blood index (BBI) for <it>An. arabiensis </it>was significantly higher for populations collected indoors (71.8%), than populations collected outdoors (41.3%), but the human blood index (HBI) did not differ significantly between the two populations. In contrast, BBI for indoor collected <it>An. funestus </it>(51.4%) was significantly lower than for outdoor collected populations (78.0%) and the HBI was significantly higher indoors (28.7%) than outdoors (2.4%). Anthropophily of <it>An. funestus </it>was lowest within the rice scheme, moderate in unplanned rice agro-ecosystem, and highest within the non-irrigated agro-ecosystem. Anthropophily of <it>An. arabiensis </it>was significantly higher in the non-irrigated agro-ecosystem than in the other agro-ecosystems.</p> <p>Conclusion</p> <p>These findings suggest that rice cultivation has an effect on host choice by <it>Anopheles </it>mosquitoes. The study further indicate that zooprophylaxis may be a potential strategy for malaria control, but there is need to assess how domestic animals may influence arboviruses epidemiology before adapting the strategy.</p

Fertilizer and Soil Health in Africa The Role of Fertilizer in Building Soil Health to Sustain Farming and Address Climate Change

Summary Soil health is commonly defined as the ability to generate sufficient crop yields while maintaining the future productive capacity of soils and the ecosystem services soils regulate and deliver. However, less consensus exists on indicators to assess soil health and its changes over time and space, although soil organic carbon (SOC) is generally acknowledged as a key indicator. In the context of this paper, soil health status is equated with SOC status. Current SOC conditions are influenced by soil properties and climate. Under smallholder farming conditions, SOC is variable and affected by past crop and soil management practices, which are influenced by farmer typology. Although SOC content under cropland is a maximum of 60-70% of that under natural vegetation, there is substantial scope to increase it in smallholder farming conditions. A conceptual framework relating to fertilizer, crop productivity, and soil health is presented here. While fertilizer application commonly results in a substantial increase in crop yield at various scales, a key indicator of fertilizer use, agronomic efficiency (AE), is often observed to be lower than relatively easily achievable values under well-managed conditions, caused by a diversity of factors. Low AE values do not necessarily result in greater greenhouse gas (GHG) emissions because of the low fertilizer application rates in sub-Saharan Africa (SSA), though increases in GHG emissions are likely with increases in fertilizer use. Crop response to organic inputs is substantially lower although organic inputs increase SOC content, which usually results in greater AE values relative to sole application of fertilizer. Increases in crop productivity are associated with increases in SOC, though the relationship is weak and efforts besides fertilizer application itself are required. That said, N(PK) fertilizer has had a positive effect on SOC in most parts of the world except SSA, an observation corroborated by an analysis of past and ongoing long-term experiments, likely related to the low and erratic use of fertilizer in the region. While fertilizer use can be an entry point to increasing soil health, this will not likely happen on degraded soils where responses to fertilizer are limited. In such cases, investments to rehabilitate degraded soils should come first. Several approaches can be followed to determine best fertilizer recommendations, while recognizing nutrients needs by crops and soil-specific properties. Site-specificity commonly requires an assessment of the soil fertility status of a particular field, and analytical tools now allow for the development of locally relevant recommendations at scale with some early successes. While organic inputs do positively impact SOC, attractive options to increase organic inputs in smallholder farming systems are limited and mostly related to in-situ production, with an important emphasis on multi-purpose legumes. Climate adaptation is facilitated by healthy Fertilizer and Soil Health in Africa 2 soils and requires fertilizer to be combined with other crop, soil, and water management practices (Wortmann and Stewart, 2021). While low yields are linked to the ecological yield gap, whereby the potential productivity of crops is set by biological factors, input and output prices determine the economic yield gap, which is usually quite lower than the former because of unfavourable ratio of fertilizer prices to crop product prices. Even though profitability is a key driver of impact, many other factors affect the adoption of appropriate fertilizer and soil health recommendations, including farmers’ production objectives, resource endowment, land tenure, and access to markets. A main bottleneck in engaging smallholder farmers in soil health-restoring practices is the relatively large amount of time such practices take to deliver benefits that are visible to farmers. In the absence of incentive programs, farmers require short-term benefits, generated within their farming systems. Furthermore, associated advice on complementary practices to fertilizer use increases the complexity of information to be conveyed to farmers. Scaling models have moved toward the delivery of bundled services, often digitally enabled, to address challenges with communicating complex information and the necessary complementary crop and soil management practices. Targeted policy interventions can support the delivery of broad digitally enabled fertilizer management recommendations and the creation of conditions that enable smallholder farmers to implement these recommendations at scale. A number of recommendations have been generated from the scientific information, covered under the following headings: (1) key elements of a Fertilizer and Soil Health Action Plan; (2) development of quantitative indicators and targets of soil health; (3) addressing climate change requires choices; (4) incentivizing farmers; (5) soil health investments, which require localized actions (think global, act local); and (6) not only fertilizers, but also auxiliary interventions, as defined by the Integrated Soil Fertility Management (ISFM) approach. Action is needed today to reverse the downward spiral of low and inefficient fertilizer use, resulting in low yields and declining soil health

In silico characterisation of putative Plasmodium falciparum vaccine candidates in African malaria populations.

Genetic diversity of surface exposed and stage specific Plasmodium falciparum immunogenic proteins pose a major roadblock to developing an effective malaria vaccine with broad and long-lasting immunity. We conducted a prospective genetic analysis of candidate antigens (msp1, ama1, rh5, eba175, glurp, celtos, csp, lsa3, Pfsea, trap, conserved chrom3, hyp9, hyp10, phistb, surfin8.2, and surfin14.1) for malaria vaccine development on 2375 P. falciparum sequences from 16 African countries. We described signatures of balancing selection inferred from positive values of Tajima's D for all antigens across all populations except for glurp. This could be as a result of immune selection on these antigens as positive Tajima's D values mapped to regions with putative immune epitopes. A less diverse phistb antigen was characterised with a transmembrane domain, glycophosphatidyl anchors between the N and C- terminals, and surface epitopes that could be targets of immune recognition. This study demonstrates the value of population genetic and immunoinformatic analysis for identifying and characterising new putative vaccine candidates towards improving strain transcending immunity, and vaccine efficacy across all endemic populations

Key genetic variants associated with variation of milk oligosaccharides from diverse human populations

Human milk oligosaccharides (HMO), the third most abundant component of human milk, are thought to be important contributors to infant health. Studies have provided evidence that geography, stage of lactation, and Lewis and secretor blood groups are associated with HMO profile. However, little is known about how variation across the genome may influence HMO composition among women in various populations. In this study, we performed genome-wide association analyses of 395 women from 8 countries to identify genetic regions associated with 19 different HMO. Our data support FUT2 as the most significantly associated (P < 4.23-9 to P < 4.5-70) gene with seven HMO and provide evidence of balancing selection for FUT2. Although polymorphisms in FUT3 were also associated with variation in lacto-N-fucopentaose II and difucosyllacto-N-tetrose, we found little evidence of selection on FUT3. To our knowledge, this is the first report of the use of genome-wide association analyses on HMO

The insecticide resistance status of malaria vectors in the Mekong region

<p>Abstract</p> <p>Background</p> <p>Knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes. Malaria transmission in the Mekong region is mainly concentrated in forested areas along the country borders, so that decisions on insecticide use should ideally be made at regional level. Consequently, cross-country monitoring of insecticide resistance is indispensable to acquire comparable baseline data on insecticide resistance.</p> <p>Methods</p> <p>A network for the monitoring of insecticide resistance, MALVECASIA, was set up in the Mekong region in order to assess the insecticide resistance status of the major malaria vectors in Cambodia, Laos, Thailand, and Vietnam. From 2003 till 2005, bioassays were performed on adult mosquitoes using the standard WHO susceptibility test with diagnostic concentrations of permethrin 0.75% and DDT 4%. Additional tests were done with pyrethroid insecticides applied by the different national malaria control programmes.</p> <p>Results</p> <p><it>Anopheles dirus s.s</it>., the main vector in forested malaria foci, was susceptible to permethrin. However, in central Vietnam, it showed possible resistance to type II pyrethroids. In the Mekong delta, <it>Anopheles epiroticus </it>was highly resistant to all pyrethroid insecticides tested. It was susceptible to DDT, except near Ho Chi Minh City where it showed possible DDT resistance. In Vietnam, pyrethroid susceptible and tolerant <it>Anopheles minimus s.l</it>. populations were found, whereas <it>An. minimus s.l</it>. from Cambodia, Laos and Thailand were susceptible. Only two <it>An. minimus s.l</it>. populations showed DDT tolerance. <it>Anopheles vagus </it>was found resistant to DDT and to several pyrethroids in Vietnam and Cambodia.</p> <p>Conclusion</p> <p>This is the first large scale, cross-country survey of insecticide resistance in <it>Anopheles </it>species in the Mekong Region. A unique baseline data on insecticide resistance for the Mekong region is now available, which enables the follow-up of trends in susceptibility status in the region and which will serve as the basis for further resistance management. Large differences in insecticide resistance status were observed among species and countries. In Vietnam, insecticide resistance was mainly observed in low or transmission-free areas, hence an immediate change of malaria vector control strategy is not required. Though, resistance management is important because the risk of migration of mosquitoes carrying resistance genes from non-endemic to endemic areas. Moreover, trends in resistance status should be carefully monitored and the impact of existing vector control tools on resistant populations should be assessed.</p