Grace Adira Murilla

Biotechnology Research Institute - KALRO · Chemotherapy

Aims: To identify viable phytomedicines traditionally employed for the treatment of malaria in Kenya that could be developed into antimalarial agents. Study Design: Quantitative analysis of antiplasmodial activities and brine shrimp bioassays were carried out using standard procedures. The experiment was set in duplicate for each concentration of t...

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clini...

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clini...

Savannah tsetse flies avoid flying toward tsetse fly-refractory waterbuck ( Kobus defassa ) mediated by a repellent blend of volatile compounds in their body odor comprised of δ-octalactone, geranyl acetone, phenols (guaiacol and carvacrol), and homologues of carboxylic acids (C 5 -C 10 ) and 2-alkanones (C 8 -C 13 ). However, although the blends o...

Vector control is an effective strategy for reducing vector‐borne disease transmission, but requires knowledge of vector habitat use and dispersal patterns. Our goal was to improve this knowledge for the tsetse species Glossina pallidipes, a vector of human and animal African trypanosomiasis, which are diseases that pose serious health and socioeco...

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focu...

Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpi...

Previous comparison of the body odors of tsetse-refractory waterbuck and those of tsetse-attractive ox and buffalo showed that a blend of 15 EAG-active compounds specific to waterbuck, including C5–C10 straight chain carboxylic acid homologues, methyl ketones (C8–C12 straight chain homologues and geranyl acetone), phenols (guaiacol and carvacrol) a...

Glossina pallidipes is the main vector of animal African trypanosomiasis and a potential vector of human African trypanosomiasis in eastern Africa where it poses a large economic burden and public health threat. Vector control efforts have succeeded in reducing infection rates, but recent resurgence in tsetse fly population density raises concerns...

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focu...

Background: Phenotypic and morphological characteristics distinguishing from bloodstream form (BSF) and central nervous system (CNS) of Trypanosoma brucei rhodesiense (Tbr) are poorly understood. Method: To identify these distinguishing characteristics, we separately infected four donor mice with each of five Tbr isolates (KETRI 2537/3537/2656/3459...

Nanoparticles (NPs) have gained importance in addressing drug delivery challenges across biological barriers. Here, we reformulated pentamidine, a drug used to treat Human African Trypanosomiasis (HAT) in polymer based nanoparticles and liposomes and compared their capability to enhance pent-amidine penetration across blood brain barrier (BBB). Siz...

Background The tsetse fly (Glossina sp.) midgut is colonized by maternally transmitted and environmentally acquired bacteria. Additionally, the midgut serves as a niche in which pathogenic African trypanosomes reside within infected flies. Tsetse’s bacterial microbiota impacts many aspects of the fly’s physiology. However, little is known about the...

The tsetse fly Glossina pallidipes, the major vector of the parasite that causes animal African trypanosomiasis in Kenya, has been subject to intense control measures with only limited success. The G. pallidipes population dynamics and dispersal patterns that underlie limited success in vector control campaigns remain unresolved, and knowledge on g...

Background: The tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense's developmental program within...

This study assessed the virulence of Trypanosoma evansi , the causative agent of camel trypanosomiasis (surra), affecting mainly camels among other hosts in Africa, Asia and South America, with high mortality and morbidity. Using Swiss white mice, we assessed virulence of 17 T. evansi isolates collected from surra endemic countries. We determined p...

Background Tsetse flies (Glossina spp.) are the prominent vector of African trypanosome parasites (Trypanosoma spp.) in sub-Saharan Africa, and Glossina pallidipes is the most widely distributed species in Kenya. This species displays strong resistance to infection by parasites, which are typically eliminated in the midgut shortly after acquisition...

Tsetse flies (Glossina spp.) transmit parasitic African trypanosomes (Trypanosoma spp.), including Trypanosoma congolense, which causes animal African trypanosomiasis (AAT). AAT detrimentally affects agricultural activities in sub-Saharan Africa and has negative impacts on the livelihood and nutrient availability for the affected communities. After...

Sheet 1. Genes with enriched expression in the proboscis (PB) compared to midgut [47] and whole head compared between uninfected PB and PB-infected with trypanosomes. The genes preferentially expressed in the PB (PB-enriched) was obtained by comparing expression of individual genes from tissues of uninfected flies. Sheet 2. Functional classificatio...

RNA-seq analysis comparing uninfected proboscis and those infected with trypanosomes from the complete transcriptome. (XLSX)

Immune-associated genes with differential expression based on RNA-seq analysis comparing uninfected PB and PB infected with trypanosomes. (XLSX)

An overview of G. m. morsitans proboscis RNA-transcriptome. (A) The total number of PB RNA-seq reads after quality control measures. (B) Proportion of reads that mapped per transcript. (C) Number of transcripts preferentially expressed in the PB (PB-enriched dataset) relative to the whole head and whole midgut transcriptomes. aPB-Proboscis—Trypanos...

Microscopic illustration of tsetse’s proboscis after Alexa Fluor 488 Phalloidin staining. (A, B and C) Tsetse’s labrum at its site of attachment to the thecal bulb, after removing the labium. The shape and general structure is observed by light microscopy, and muscles are fluorescence green after staining with phalloidin (dyes actin). Shown are the...

Graphical representation on transcript abundance of genes encoding mechanoreceptors. The heat map was generated by plotting the normalized RPKM values (Log2 transformed) of individual transcript from uninfected fly tissues, clustered using euclidean distance calculation and ward.D clustering methods. PB proboscis, WH whole head, WMG whole midgut, S...

Primers utilized for tsetse fly PB validation. (DOCX)

Genes encoding transmembrane and/or secreted proteins. (XLSX)

Validation of tsetse RNA-seq results with qPCR. (DOCX)

Background Glossina pallidipes is a major vector of both Human and Animal African Trypanosomiasis (HAT and AAT) in Kenya. The disease imposes economic burden on endemic regions in Kenya, including south-western Kenya, which has undergone intense but unsuccessful tsetse fly control measures. We genotyped 387 G. pallidipes flies at 13 microsatellite...

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and...

Diagnostic PCR for the GCT/Ala281 deletion in F1FO-ATP synthase subunit γ in T. evansi type A. Shown are nucleotides 1–859 (GCT deletion) and 1–863 (‘wild type’), respectively, of gene TevSTIB805.10.220 / Tb427.10.180 (systematic TriTrypDB.org IDs). Primer combination F1/R1 will give a 855-bp amplicon if the deletion is present. Primer combination...

STRUCTURE v2.3.4 [51] plot of individual assignments with K values of 2 through 7. Each vertical bar represents a strain’s probability of assignment to one of K genetic clusters, with T. brucei (Tb) strains on the left (light gray horizontal bar) and T. evansi (Tev) strains on the right (dark gray horizontal bar). Individuals with 100% probability...

PCR primers used in microsatellite marker amplification, with general information about the motif, size range in bp (size), chromosome location (location), and source of the protocol used. (DOCX)

Among-cluster genetic differentiation (FST) among each STRUCTURE-defined [51] genetic cluster, using only strains with Q values >0.80 (S3 Table): (A) all strains, (B) T. brucei (Tb) strains only, and (C) T. evansi (Tev) strains only. Pairwise FST (below diagonal) was calculated in ARLEQUIN v.3.2 [59] with Wright’s statistics [60], following the var...

Distance tree based on 15 microsatellite markers and Reynolds et al (1983) distances using the UPGMA method implemented in the R package, “PopPR” v2.3 [54, 55]. Support values are shown on nodes only for values above 50% and are based on 1000 bootstrap replicates. Terminal tips identify the strains (Table 1 and S1 Table) and are color coded accordi...

STRUCTURE v2.3.4 [51] plot of delta K for K values of 2 to 9 based on 20 runs each performed with a burn-in of 5,000 and a total of 250,000 iterations. Although K = 2 had the highest delta K and thus explained the highest hierarchical level in the data, a K value of 7 was the next hierarchical level with a peak in delta K, and was able to distingui...

Summary of pairwise Reynolds (1983) genetic distances computed in the R package. “PopPR” v2.3.0 [54, 55] between strains belonging to the same or different STRUCTURE-defined clusters as outlier box-plots color coded according to legend to the left. Boxes and whiskers on each box-plot represent the minimum, 1st quartile, 3rd quartile, and maximum di...

Sample details of strains from previous studies showing sample ID, publication, taxon, kDNA, host of isolation, locality of origin and year of isolation, n/a indicates no history found on the year of isolation. (DOCX)

Assignment scores from STRUCTURE v2.3.4 [51] clustering analysis with K = 7 showing sample ID, taxon, genetic cluster “a-g” (Fig 2) if probability of assignment (Q) above or equal to 0.8, or "uncertain" if Q < 0.8 for each strain of (A) Trypanosoma brucei brucei (Tbb) or T. b. rhodesiense (Tbr), and (B) T. evansi (Tev). (DOCX)

Within-cluster distance using STRUCTURE-based [51] genetic clusters including strains with Q values > 0.80 (S3 Table) for (A) all strains regardless of taxonomy, (B) T. brucei (Tb) strains, and (C) T. evansi (Tev) strains. Number of pairwise between-strain comparisons (N pairs), mean Reynolds (1983) [56] distance (mean distance) estimated in the R...

Summary of differences in within-cluster Reynolds [56] distance of STRUCTURE-defined clusters based on analysis of variance (ANOVA, p-value < 0.0001), and the Tukey-Kramer HSD test performed in JMP v11.2 (SAS Institute Inc., Cary, NC, USA, 1989–2012), using only the 86 strains with Q values >0.80 (S3 Table): (A) Ordered difference report between cl...

African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.). In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolen...

[This corrects the article DOI: 10.1371/journal.pntd.0004421.].

Background and purpose: Medicinal plants including the prickly pear cactus have been reported to modulate blood sugar levels. Extracts of prickly pear cactus have been used in various parts of the world to manage diabetes mellitus. However the cactus is viewed as a weed in Kenya. The current study therefore aimed at evaluating the efficacy of prick...

For decades, odour-baited traps have been used for control of tsetse flies (Diptera; Glossinidae), vectors of African trypanosomes. However, differential responses to known attractants have been reported in different Glossina species, hindering establishment of a universal vector control tool. Availability of full genome sequences of five Glossina...

Alignment files of the amino acid sequences. Multiple sequence alignments of five Glossina species, Drosophila melanogaster, Anopheles gambaie, Musca domestica and Ceratitis capitata used in construction of phylogenies of the chemosensory gene. (ZIP)

Alignment of IRs amino acid sequences showing conserved residues that constitute the pore region. (PDF)

Metadata for genes annotated in four species of Glossina. Metadata for each protein chemosensory family is contained in a separate sheet. CSPs—sheet 1, SNMPs—sheet 2, GRs –sheet3, OBPs –sheet 4, ORs –sheet 5 and IRs –sheet 6. For every sequence, the following data is provided in columns A-G of each sheet: Gene name, VectorBase identifier, scaffold...

VectorBase web Apollo screenshots. Screenshots illustrating gene structure and tandem arrangement of selected chemosensory genes. Four copies of Obp83a (part A) thought to be olfactory specific in GlossinaTwo Or7a homologs (part B) and two Or56a homologs (part C). (PDF)

Tsetse chemosensory genes identified as having signatures of positive selection by codon-based alignment methods MEME and PARRIS in Datamonkey analysis. (PDF)

Glossina chemosensory gene loci identified to have signatures by PAML analysis using the M8-M8a model. lnL M8 is the likelihood of the experimental model (M8), lnL M8a is the likelihood of the null model (M8a), ΔLRT is the Likelihood Ratio Test = 2*(lnL M8- lnL M8a), w1M8 is the ratio of Non-synonymous to synonymous mutations (dN/dS) predicted unde...

Multiple alignment of Obp19 and Obp56i from Glossina, Obp16-20 from M. domestica and Obp56i from D. melanogaster. Variation of amino acids between conserved cysteine(s) C3 and C4 in Obp56i and Obp19 from Glossina. Their homologs in M. domestica and D. melanogaster appear more conserved around the same region. (PDF)