Antimicrobial resistance profiles of salmonella spp. and escherichia coli isolated from fresh nile tilapia (oreochromis niloticus) fish marketed for human consumption.

BACKGROUND: Salmonella spp. and pathogenic strains of Escherichia coli are among the major foodborne zoonotic pathogens. These bacterial pathogens cause human illnesses characterized by hemorrhagic colitis, vomiting, nausea, and other agent-related symptoms. The increasing occurrence of antimicrobial resistance in these pathogens is also a serious public health concern globally. Regular surveillance of phenotypes and genotypes of Salmonella spp. and Escherichia coli from animal-derived foods is necessary for effective reduction and control of these foodborne pathogens. This study was conducted to assess the occurrence, antimicrobial resistance, virulence genes and genetic diversity of Salmonella spp. and E. coli isolates from fresh Nile tilapia obtained from retail markets in Nairobi, Kenya. METHODS: A total of 68 fresh Nile tilapia fish samples were collected from retail markets and used for isolation of Salmonella spp. and E. coli. Antimicrobial susceptibilities of the isolates weretested by Kirby-Bauer agar disc diffusion method. According to the antimicrobial resistance profiles, the multi-drug resistant isolates were identified by 16 S rRNA sequencing and phylogenetic analysis using the Bayesian inference method. The MDR Salmonella spp. and E. coli isolates were subjected to PCR-based screening for the detection virulence and antibiotic resistance genes. RESULTS: The prevalence of contamination of the fish samples with Salmonella spp. and E.coli was 26.47% and 35.29% respectively. Overall phenotypic resistance among the Salmonella spp. ranged from 5.5% for ceftazidime, chloramphenicol, meropenem, nitrofurantoin and streptomycin and 22.2% for penicillin-G. For E. coli phenotypic resistance ranged from 4.2% for ceftazidime and chloramphenicol and 25% for rifampicin. Multi-drug resistance was observed in three Salmonella spp. and two E. coli isolates. Results of 16 S rRNA sequences, sequence alignment and phylogenic trees confirmed the identified MDR isolates as S. typhymurium WES-09, S. typhymurium MAK-22, S. typhimurium EMB-32 and E. coli MAK-26 and E. coli LAN-35. The presence of antibiotic-resistance genes belonging to ß-lactamases, tetracycline, sulfonamide, trimethoprim and aminoglycosides-resistant genes were detected in all the identified MDR isolates. CONCLUSIONS: The findings from this study indicate that Nile tilapia (Oreochromis niloticus) sold in retail markets can acts as reservoirs of Salmonella spp. and E. coli pathogens linked to human disease, some of which were multidrug resistance to critically important antimicrobials. Both microorganisms are of zoonotic significance and represent a significant public health risk to the society.

Antimalarial activity assay of artesunate-3-chloro-4(4-chlorophenoxy) aniline in vitro and in mice models.

The global spread of multi-drug resistant P. falciparum, P. vivax, and P. malariae strains and absence of long-term effective vaccine makes chemotherapy the mainstay of malaria control strategies in endemic settings. The Mossman's assay and the Organization for Economic Co-operation and Development (OECD), 2001 guideline 423, were used to determine the cytotoxicity and acute oral toxicity of a novel hybrid drug, artesunate-3-Chloro-4(4-chlorophenoxy) aniline (ATSA), in vitro and in vivo, respectively. A modified Desjardins method was used to screen for antiplasmodial activity using P. falciparum (3D7 and W2) strains in vitro. The Peter's 4-day suppressive tests (4DTs) was used to evaluate the in vivo antimalaria activity using P. berghei ANKA strain, lumefantrine resistant (LuR), and piperaquine resistant (PQR) P. berghei lines. In silico prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles was assayed using PreADMET online prediction tool. The reference drug in all experiments was artesunate (ATS). Statistical significance between ATSA's activities in treated and control mice was evaluated by one-way analysis of variance (ANOVA). Results show that inhibitory concentrations-50 (IC50) of ATSA is 11.47 ± 1.3 (3D7) and 1.45 ± 0.26 (W2) against 4.66 ± 0.93 (3D7) and 0.60 ± 0.15 (W2) ng/ml of ATS with a selective index of 2180.91(3D7) and a therapeutic index (TI) of > 71). No mortalities were observed in acute oral toxicity assays and mean weight differences for test and controls were statistically insignificant (P > 0.05). The in vivo activity of ATSA was above 40% with effective dosage-50 (ED50) of 4.211, 2.601, and 3.875 mg/kg body weight against P. berghei ANKA, LuR, and PQR lines, respectively. The difference between treated and control mice was statistically significant (P < 0.05). ATSA has high intestinal absorption (HIA) > 95% and has medium human ether-a-go-go related gene (hERG) K+ channel inhibition risks. Preclinical and clinical studies on ATSA are recommended to evaluate its value in developing novel drugs for future management of multi-drug resistant malaria parasites.

Prevalence of pfdhfr and pfdhps mutations in Plasmodium falciparum associated with drug resistance among pregnant women receiving IPTp-SP at Msambweni County Referral Hospital, Kwale County, Kenya.

BACKGROUND: Prevention and treatment of malaria during pregnancy is crucial in dealing with maternal mortality and adverse fetal outcomes. The World Health Organization recommendation to treat all pregnant women with sulfadoxine-pyrimethamine (SP) through antenatal care structures was implemented in Kenya in the year 1998, but concerns about its effectiveness in preventing malaria in pregnancy has arisen due to the spread of SP resistant parasites. This study aimed to determine the prevalence of SP resistance markers in Plasmodium falciparum parasites isolated from pregnant women seeking antenatal care at Msambweni County Referral Hospital, located in coastal Kenya, between the year 2013 and 2015. METHODS: This hospital-based study included 106 malaria positive whole blood samples for analysis of SP resistance markers within the Pfdhfr gene (codons 51, 59 and 108) and Pfdhps gene (codons 437 and 540). The venous blood collected from all pregnant women was tested for malaria via light microscopy, then the malaria positive samples were separated into plasma and red cells and stored in a - 86° freezer for further studies. Archived red blood cells were processed for molecular characterization of SP resistance markers within the Pfdhfr and Pfdhps genes using real time PCR platform and Sanger sequencing. RESULTS: All samples had at least one mutation in the genes associated with drug resistance; polymorphism prevalence of Pfdhfr51I, 59R and 108N was at 88.7%, 78.3% and 93.4%, respectively, while Pfdhps polymorphism accounted for 94.3% and 91.5% at 437G and 540E, respectively. Quintuple mutations (at all the five codons) conferring total SP resistance had the highest prevalence of 85.8%. Quadruple mutations were observed at a frequency of 10.4%, and 24.5% had a mixed outcome of both wildtype and mutant genotypes in the genes of interest. CONCLUSION: The data suggest a high prevalence of P. falciparum genetic variations conferring resistance to SP among pregnant women, which may explain reduced efficacy of IPTp treatment in Kenya. There is need for extensive SP resistance profiling in Kenya to inform IPTp drug choices for successful malaria prevention during pregnancy.

Combination of Linkage Mapping, GWAS, and GP to Dissect the Genetic Basis of Common Rust Resistance in Tropical Maize Germplasm.

Common rust (CR) caused by Puccina sorghi is one of the destructive fungal foliar diseases of maize and has been reported to cause moderate to high yield losses. Providing CR resistant germplasm has the potential to increase yields. To dissect the genetic architecture of CR resistance in maize, association mapping, in conjunction with linkage mapping, joint linkage association mapping (JLAM), and genomic prediction (GP) was conducted on an association-mapping panel and five F3 biparental populations using genotyping-by-sequencing (GBS) single-nucleotide polymorphisms (SNPs). Analysis of variance for the biparental populations and the association panel showed significant genotypic and genotype x environment (GXE) interaction variances except for GXE of Pop4. Heritability (h2) estimates were moderate with 0.37-0.45 for the individual F3 populations, 0.45 across five populations and 0.65 for the association panel. Genome-wide association study (GWAS) analyses revealed 14 significant marker-trait associations which individually explained 6-10% of the total phenotypic variances. Individual population-based linkage analysis revealed 26 QTLs associated with CR resistance and together explained 14-40% of the total phenotypic variances. Linkage mapping revealed seven QTLs in pop1, nine QTL in pop2, four QTL in pop3, five QTL in pop4, and one QTL in pop5, distributed on all chromosomes except chromosome 10. JLAM for the 921 F3 families from five populations detected 18 QTLs distributed in all chromosomes except on chromosome 8. These QTLs individually explained 0.3 to 3.1% and together explained 45% of the total phenotypic variance. Among the 18 QTL detected through JLAM, six QTLs, qCR1-78, qCR1-227, qCR3-172, qCR3-186, qCR4-171, and qCR7-137 were also detected in linkage mapping. GP within population revealed low to moderate correlations with a range from 0.19 to 0.51. Prediction correlation was high with r = 0.78 for combined analysis of the five F3 populations. Prediction of biparental populations by using association panel as training set reveals positive correlations ranging from 0.05 to 0.22, which encourages to develop an independent but related population as a training set which can be used to predict diverse but related populations. The findings of this study provide valuable information on understanding the genetic basis of CR resistance and the obtained information can be used for developing functional molecular markers for marker-assisted selection and for implementing GP to improve CR resistance in tropical maize.

In vitro selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemisinin.

BACKGROUND: Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance. METHODS: Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast. RESULTS: Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene. DISCUSSION: This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria. CONCLUSION: Artemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Phytochemical Screening and GC-MS Analysis of Methanolic and Aqueous Extracts of Ocimum kilimandscharicum Leaves.

Kenyans have long utilized Ocimum kilimandscharicum, an East African permanent evergreen plant, to treat measles, stomachaches, diarrhea, mosquito bites (anti-insect), congested chest, cough, and colds. Using conventional qualitative and quantitative techniques, this study was done to identify the secondary metabolites in O. kilimandscharicum leaf extracts. The chemical content of the crude extracts from the leaves of O. kilimandscharicum has also been investigated and characterized using gas chromatography-mass spectrometry (GC-MS). By using a 1:20 dilution in methanol, in cold maceration, a fine powder of O. kilimandscharicum was first extracted then filtered and concentrated after 72 h utilizing a rotary evaporator. By using also a 1:20 dilution in water at 80 °C, a fine powder of O. kilimandscharicum was extracted and then filtrated and lyophilized 1 h later. Each extract underwent further gas chromatography-mass spectrometry testing. We found that both extracts contain secondary metabolites such as alkaloids, phenolics, flavonoids, saponins, and tannins. However, the overall amount of phytochemicals in each solvent varied significantly. Total phenolics contents (TPCs) were 5.6 ± 1.20 and 10.8 ± 1.00 mg, total flavonoid contents (TFCs) were 8.2 ± 0.4 and 39.6 ± 2.2 mg, total tannin contents (TTCs) were 0 ± 0.00 and 10.5 ± 0.4 mg, the total alkaloid content (TAC) was 49.2 ± 0.40%, and the total saponin content (TSC) was 38 ± 2.00%. Additionally the gas chromatography-mass spectrometry, revealed a number of high- and low-molecular-weight bioactive molecules at various concentrations for each extract. We also found an inhibitory effect on adhP and chbR gene expression of Staphylococcus aureus and Salmonella choleraesuius, respectively. Hence, these chemicals could potentially have a biological and pharmacological significance. Therefore, the discovery of many physiologically active chemicals in the leaf extracts of O. kilimandscharicum justifies future biological and pharmaceutical research.

Limited genetic variations of the Rh5-CyRPA-Ripr invasion complex in Plasmodium falciparum parasite population in selected malaria-endemic regions, Kenya.

The invasion of human erythrocytes by Plasmodium falciparum merozoites requires interaction between parasite ligands and host receptors. Interaction of PfRh5-CyRPA-Ripr protein complex with basigin, an erythrocyte surface receptor, via PfRh5 is essential for erythrocyte invasion. Antibodies raised against each antigen component of the complex have demonstrated erythrocyte invasion inhibition, making these proteins potential blood-stage vaccine candidates. Genetic polymorphisms present a significant challenge in developing efficacious vaccines, leading to variant-specific immune responses. This study investigated the genetic variations of the PfRh5 complex proteins in P. falciparum isolates from Lake Victoria islands, Western Kenya. Here, twenty-nine microscopically confirmed P. falciparum field samples collected from islands in Lake Victoria between July 2014 and July 2016 were genotyped by whole genome sequencing, and results compared to sequences mined from the GenBank database, from a study conducted in Kilifi, as well as other sequences from the MalariaGEN repository. We analyzed the frequency of polymorphisms in the PfRh5 protein complex proteins, PfRh5, PfCyRPA, PfRipr, and PfP113, and their location mapped on the 3D protein complex structure. We identified a total of 58 variants in the PfRh5 protein complex. PfRh5 protein was the most polymorphic with 30 SNPs, while PfCyRPA was relatively conserved with 3 SNPs. The minor allele frequency of the SNPs ranged between 1.9% and 21.2%. Ten high-frequency alleles (>5%) were observed in PfRh5 at codons 147, 148, 277, 410, and 429 and in PfRipr at codons 190, 255, 259, and 1003. A SNP was located in protein-protein interaction region C203Y and F292V of PfRh5 and PfCyRPA, respectively. Put together, this study revealed low polymorphisms in the PfRh5 invasion complex in the Lake Victoria parasite population. However, the two mutations identified on the protein interaction regions prompts for investigation on their impacts on parasite invasion process to support the consideration of PfRh5 components as potential malaria vaccine candidates.

Reducing false negative reads in RFID data streams using an adaptive sliding-window approach.

Unreliability of the data streams generated by RFID readers is among the primary factors which limit the widespread adoption of the RFID technology. RFID data cleaning is, therefore, an essential task in the RFID middleware systems in order to reduce reading errors, and to allow these data streams to be used to make a correct interpretation and analysis of the physical world they are representing. In this paper we propose an adaptive sliding-window based approach called WSTD which is capable of efficiently coping with both environmental variation and tag dynamics. Our experimental results demonstrate the efficacy of the proposed approach.

Preliminary In Vivo Evidence of Oral Selenium Supplementation as a Potentiating Agent on a Vector-Based COVID-19 Vaccine in BALB/c Mice.

Evidence of efficacy and toxicity of oral selenium supplementation in vaccine administration against severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in mice models is scarce. In this study, 4 × 109 virus particles (40 µL) dose of Janssen COVID-19 intramuscular injection vaccine was supplemented with a commercial selenium supplement and sodium selenite orally in BALB/c mice (N = 18). Qualitative determination of anti-spike IgG antibody response using indirect Enzyme-Linked Immunosorbent Assay (ELISA) showed significant (p ≤ 0.001) increase in anti-spike IgG antibody response for mice groups immunized with vaccine and supplemented selenium. Furthermore, cytokine profiling using real-time quantitative polymerase chain reaction also showed an increase in IL-6 and IL-10 mRNA levels normalized using hypoxanthine phosphoribosyl transferase 1 (Hprt1) and glyceraldehyde 3-phosphate dehydrogenase (Gadph) housekeeping genes. There was no statistical significance (p < 0.465) among treated and untreated groups for alanine transaminase (ALT), aspartate transaminase (AST), urea, and creatinine parameters. The study presents preliminary findings and suggests that supplementing Janssen COVID-19 vaccines with selenium can generate more robust immune responses.

Molecular detection of novel Anaplasma sp . and zoonotic hemopathogens in livestock and their hematophagous biting keds (genus Hippobosca) from Laisamis, northern Kenya.

Background: Livestock are key sources of livelihood among pastoral communities. Livestock productivity is chiefly constrained by pests and diseases. Due to inadequate disease surveillance in northern Kenya, little is known about pathogens circulating within livestock and the role of livestock-associated biting keds (genus Hippobosca) in disease transmission. We aimed to identify the prevalence of selected hemopathogens in livestock and their associated blood-feeding keds. Methods: We randomly collected 389 blood samples from goats (245), sheep (108), and donkeys (36), as well as 235 keds from both goats and sheep (116), donkeys (11), and dogs (108) in Laisamis, Marsabit County, northern Kenya. We screened all samples for selected hemopathogens by high-resolution melting (HRM) analysis and sequencing of PCR products amplified using primers specific to the genera: Anaplasma, Trypanosoma, Clostridium, Ehrlichia, Brucella, Theileria, and Babesia. Results: In goats, we detected Anaplasma ovis (84.5%), a novel Anaplasma sp. (11.8%), Trypanosoma vivax (7.3%), Ehrlichia canis (66.1%), and Theileria ovis (0.8%). We also detected A. ovis (93.5%), E. canis (22.2%), and T. ovis (38.9%) in sheep. In donkeys, we detected ' Candidatus Anaplasma camelii' (11.1%), T. vivax (22.2%), E. canis (25%), and Theileria equi (13.9%). In addition, keds carried the following pathogens; goat/sheep keds - T. vivax (29.3%) , Trypanosoma evansi (0.86%), Trypanosoma godfreyi (0.86%), and E. canis (51.7%); donkey keds - T. vivax (18.2%) and E. canis (63.6%); and dog keds - T. vivax (15.7%), T. evansi (0.9%), Trypanosoma simiae (0.9%) , E. canis (76%), Clostridium perfringens (46.3%), Bartonella schoenbuchensis (76%), and Brucella abortus (5.6%). Conclusions: We found that livestock and their associated ectoparasitic biting keds carry a number of infectious hemopathogens, including the zoonotic B. abortus. Dog keds harbored the most pathogens, suggesting dogs, which closely interact with livestock and humans, as key reservoirs of diseases in Laisamis. These findings can guide policy makers in disease control.

Screening for antifolate and artemisinin resistance in Plasmodium falciparum dried-blood spots from three hospitals of Eritrea.

Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea's population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea. Methods: Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions. Results: Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate. Conclusions: The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples.

Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.

Genetic Dissection of Resistance to Gray Leaf Spot by Combining Genome-Wide Association, Linkage Mapping, and Genomic Prediction in Tropical Maize Germplasm.

Gray leaf spot (GLS) is one of the major maize foliar diseases in sub-Saharan Africa. Resistance to GLS is controlled by multiple genes with additive effect and is influenced by both genotype and environment. The objectives of the study were to dissect the genetic architecture of GLS resistance through linkage mapping and genome-wide association study (GWAS) and assessing the potential of genomic prediction (GP). We used both biparental populations and an association mapping panel of 410 diverse tropical/subtropical inbred lines that were genotyped using genotype by sequencing. Phenotypic evaluation in two to four environments revealed significant genotypic variation and moderate to high heritability estimates ranging from 0.43 to 0.69. GLS was negatively and significantly correlated with grain yield, anthesis date, and plant height. Linkage mapping in five populations revealed 22 quantitative trait loci (QTLs) for GLS resistance. A QTL on chromosome 7 (qGLS7-105) is a major-effect QTL that explained 28.2% of phenotypic variance. Together, all the detected QTLs explained 10.50, 49.70, 23.67, 18.05, and 28.71% of phenotypic variance in doubled haploid (DH) populations 1, 2, 3, and F3 populations 4 and 5, respectively. Joint linkage association mapping across three DH populations detected 14 QTLs that individually explained 0.10-15.7% of phenotypic variance. GWAS revealed 10 significantly (p < 9.5 × 10-6) associated SNPs distributed on chromosomes 1, 2, 6, 7, and 8, which individually explained 6-8% of phenotypic variance. A set of nine candidate genes co-located or in physical proximity to the significant SNPs with roles in plant defense against pathogens were identified. GP revealed low to moderate prediction correlations of 0.39, 0.37, 0.56, 0.30, 0.29, and 0.38 for within IMAS association panel, DH pop1, DH pop2, DH pop3, F3 pop4, and F3 po5, respectively, and accuracy was increased substantially to 0.84 for prediction across three DH populations. When the diversity panel was used as training set to predict the accuracy of GLS resistance in biparental population, there was 20-50% reduction compared to prediction within populations. Overall, the study revealed that resistance to GLS is quantitative in nature and is controlled by many loci with a few major and many minor effects. The SNPs/QTLs identified by GWAS and linkage mapping can be potential targets in improving GLS resistance in breeding programs, while GP further consolidates the development of high GLS-resistant lines by incorporating most of the major- and minor-effect genes.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein.

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds.  Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.

Effect of Indigenous and Introduced Arbuscular Mycorrhizal Fungi on Growth and Phytochemical Content of Vegetatively Propagated Prunus Africana (Hook. f.) Kalkman Provenances.

Prunus africana bark contains phytochemical compounds used in the treatment of benign prostatic hyperplasia and prostate cancer. It has been shown that this plant establishes association with arbuscular mycorrhizal fungi (AMF). AMF are involved in nutrient uptake, which may also affect plant growth and secondary metabolites composition. However, there is no information regarding the role of AMF in the growth and phytochemical content of P. africana. A pot experiment was carried out to assess the response of 8 months old vegetatively propagated P. africana seedlings inoculated with indigenous AMF collected from Mount Cameroon (MC) and Mount Manengumba (MM) in Cameroon, Malava near Kakamega (MK) and Chuka Tharaka-Nithi (CT) in Kenya. Mycorrhizal (frequency, abundance and intensity), growth (height, shoot weight, total weight, number of leaf, leaf surface) and phytochemical (total phenol, tannin and flavonoids) parameters were measured three months after growth of seedlings from two provenances (Muguga and Chuka) with the following inoculation treatments: MK, CT, MC, MM, non-sterilized soil (NS) and sterilized sand as non-inoculated control. Results showed that seedling heights were significantly increased by inoculation and associated with high root colonization (>80%) compared to non-inoculated seedlings. We also found that AMF promoted leaf formation, whereas inoculation did not have any effect on the seedling total weight. AMF inoculum from MM had a higher tannin content, while no significant difference was observed on the total phenol and flavonoid contents due to AMF inoculation. Pearson's correlation was positive between mycorrhizal parameters and the growth parameters, and negative with phytochemical parameters. This study is the first report on the effect of AMF on the growth and phytochemical in P. africana. Further investigations are necessary to determine the effect of single AMF strains to provide better understanding of the role of AMF on the growth performance and physiology of this important medicinal plant species.

In Silico Characterization and Structural Modeling of Dermacentor andersoni p36 Immunosuppressive Protein.

Ticks cause approximately $17-19 billion economic losses to the livestock industry globally. Development of recombinant antitick vaccine is greatly hindered by insufficient knowledge and understanding of proteins expressed by ticks. Ticks secrete immunosuppressant proteins that modulate the host's immune system during blood feeding; these molecules could be a target for antivector vaccine development. Recombinant p36, a 36 kDa immunosuppressor from the saliva of female Dermacentor andersoni, suppresses T-lymphocytes proliferation in vitro. To identify potential unique structural and dynamic properties responsible for the immunosuppressive function of p36 proteins, this study utilized bioinformatic tool to characterize and model structure of D. andersoni p36 protein. Evaluation of p36 protein family as suitable vaccine antigens predicted a p36 homolog in Rhipicephalus appendiculatus, the tick vector of East Coast fever, with an antigenicity score of 0.7701 that compares well with that of Bm86 (0.7681), the protein antigen that constitute commercial tick vaccine Tickgard™. Ab initio modeling of the D. andersoni p36 protein yielded a 3D structure that predicted conserved antigenic region, which has potential of binding immunomodulating ligands including glycerol and lactose, found located within exposed loop, suggesting a likely role in immunosuppressive function of tick p36 proteins. Laboratory confirmation of these preliminary results is necessary in future studies.

Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection of Cryptosporidium Species in Kenyan Children Presenting with Diarrhea.

Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP) technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The test has a detection limit of 10 pg/µl (~100 oocysts/ml) indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD) LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers). Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the detection of C. hominis, C. parvum, and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.

BACKGROUND: Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available. METHODS: In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples. RESULTS: The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10-7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10-5 (~3 ng/ml) and 10-4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide. CONCLUSION: The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.

Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples.

BACKGROUND: Hookworm infection is a major concern in sub-Saharan Africa, particularly in children and pregnant women. Necator americanus and Ancylostoma duodenale are responsible for this condition. Hookworm disease is one of the Neglected tropical diseases (NTDs) that are targeted for elimination through global mass chemotherapy. To support this there is a need for reliable diagnostic tools. The conventional diagnostic test, Kato-Katz that is based on microscopic detection of parasite ova in faecal samples, is not effective due to its low sensitivity that is brought about mainly by non-random distribution of eggs in stool and day to day variation in egg output. It is tedious, cumbersome to perform and requires experience for correct diagnosis. LAMP-based tests are simple, relatively cheap, offer greater sensitivity, specificity than existing tests, have high throughput capability, and are ideal for use at the point of care. METHODS: We have developed a LAMP diagnostic test for detection of hookworm infection in faecal samples. LAMP relies on auto cycling strand displacement DNA synthesis performed at isothermal temperature by Bst polymerase and a set of 4 specific primers. The primers used in the LAMP assay were based on the second Internal Transcribed Spacer (ITS-2) region and designed using Primer Explorer version 4 Software. The ITS-2 region of the ribosomal gene (rDNA) was identified as a suitable target due to its low mutation rates and substantial differences between species. DNA was extracted directly from human faecal samples, followed by LAMP amplification at isothermal temperature of 63 °C for 1 h. Amplicons were visualized using gel electrophoresis and SYBR green dye. Both specificity and sensitivity of the assay were determined. RESULTS: The LAMP based technique developed was able to detect N. americanus DNA in faecal samples. The assay showed 100 % specificity and no cross-reaction was observed with other helminth parasites (S. mansoni, A. lumbricoides or T. trichiura). The developed LAMP assay was 97 % sensitive and DNA at concentrations as low as 0.4 fg were amplified. CONCLUSION: The LAMP assay developed is an appropriate diagnostic method for the detection of N. americanus DNA in human stool samples because of its simplicity, low cost, sensitivity, and specificity. It holds great promise as a useful diagnostic tool for use in disease control where infection intensities have been significantly reduced.

Immunization of rabbits with Glossina pallidipes tsetse fly midgut proteins: effects on the fly and trypanosome transmission.

Proteins isolated from the midgut of Glossina pallidipes were used to immunize rabbits and their efficacy as vaccine candidate(s) against the fly, and their potential to block transmission of Trypanosoma brucei rhodesiense assessed. Two fractions, detergent (DET) and aqueous (AQ) fractions were separated using a non-ionic detergent (Triton X-114) and a series of bioassay experiments carried out using serum obtained from rabbits immunized with either of the two fractions. The mortality rates of tsetse flies fed on serum from rabbits immunized with DET and AQ was 56 and 35%, respectively, as compared to 20% mortality in controls. The DET antigen(s) caused considerably higher mortality (chi(2)=1.194, P<0.05) than that on controls. These findings suggest that midgut proteins contain antigens that are lethal to tsetse flies, and are potential candidates for the development of anti-tsetse vaccine. When flies fed on serum derived from DET immunized rabbits were fed on T. b. rhodesiense infected blood, only 20% of them picked the infection. Very few flies (20%) fed on serum derived from DET immunized rabbits had infection of T. b. rhodesiense. In the control flies 45% of them had infection in the midgut with a higher and actively motile parasite load. Assessment of fecundity indicated significantly higher (chi(2)=2.117, P<0.05) larviposition for the control flies when compared to the AQ group of flies (chi(2)=1.054, P<0.05). Significant differences in abortions and pupal weights were also observed. These results suggest that midgut proteins contain antigens with potential for use in development of vaccine to block transmission of trypanosomes through tsetse.