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BackgroundAfrican Trypanosomiases threaten the life of both humans and animals. Trypanosomes are transmitted by tsetse and other biting flies. In Rwanda, the African Animal Trypanosomiasis (AAT) endemic area is mainly around the tsetse-infested Akagera National Park (NP). The study aimed to identify Trypanosoma species circulating in cattle, their genetic diversity and distribution around the Akagera NP.MethodologyA cross-sectional study was carried out in four districts, where 1,037 cattle blood samples were collected. The presence of trypanosomes was determined by microscopy, immunological rapid test VerY Diag and PCR coupled with High-Resolution Melt (HRM) analysis. A parametric test (ANOVA) was used to compare the mean Packed cell Volume (PCV) and trypanosomes occurrence. The Cohen Kappa test was used to compare the level of agreement between the diagnostic methods.FindingsThe overall prevalence of trypanosome infections was 5.6%, 7.1% and 18.7% by thin smear, Buffy coat technique and PCR/HRM respectively. Microscopy showed a low sensitivity while a low specificity was shown by the rapid test (VerY Diag). Trypanosoma (T.) congolense was found at a prevalence of 10.7%, T. vivax 5.2%, T. brucei brucei 2% and T. evansi 0.7% by PCR/HRM. This is the first report of T.evansi in cattle in Rwanda. The non-pathogenic T. theileri was also detected. Lower trypanosome infections were observed in Ankole x Friesian breeds than indigenous Ankole. No human-infective T. brucei rhodesiense was detected. There was no significant difference between the mean PCV of infected and non-infected animals (p>0.162).ConclusionsOur study sheds light on the species of animal infective trypanosomes around the Akagera NP, including both pathogenic and non-pathogenic trypanosomes. The PCV estimation is not always an indication of trypanosome infection and the mechanical transmission should not be overlooked. The study confirms that the area around the Akagera NP is affected by AAT, and should, therefore, be targeted by the control activities. AAT impact assessment on cattle production and information on the use of trypanocides are needed to help policymakers prioritise target areas and optimize intervention strategies. Ultimately, these studies will allow Rwanda to advance in the Progressive Control Pathway (PCP) to reduce or eliminate the burden of AAT.

Anaplasmosis, caused by infection with bacteria of the genus Anaplasma, is an important veterinary and zoonotic disease. Transmission by ticks has been characterized but little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence of Anaplasma spp. in camels in northern Kenya and whether the hematophagous camel ked, Hippobosca camelina, acts as a vector. Camels (n = 976) and > 10,000 keds were sampled over a three-year study period and the presence of Anaplasma species was determined by PCR-based assays targeting the Anaplasmataceae 16S rRNA gene. Camels were infected by a single species of Anaplasma, 'Candidatus Anaplasma camelii', with infection rates ranging from 63-78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10-29% of camel keds harbored 'Ca. Anaplasma camelii' acquired from infected camels during blood feeding. We determined that Anaplasma-positive camel keds could transmit 'Ca. Anaplasma camelii' to mice and rabbits via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by microscopic observation in blood smears and by PCR. Transmission of 'Ca. Anaplasma camelii' to mice (8-47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential of H. camelina as a vector of anaplasmosis. This key finding provides the rationale for establishing ked control programmes for improvement of livestock and human health.

Anopheles mosquitoes are colonized by diverse microorganisms that may impact on host biology and vectorial capacity. Eukaryotic symbionts such as fungi have been isolated from Anopheles, but whether they are stably associated with mosquitoes and transmitted transstadially across mosquito life stages or to subsequent generations remains largely unexplored. Here, we show that a Leptosphaerulina sp. fungus isolated from the midgut of An. gambiae can be stably associated with An. gambiae host and that it imposes low fitness cost when re-introduced through co-feeding. This fungus is transstadially transmitted across An. gambiae developmental stages and to their progeny. It is present in field-caught larvae and adult mosquitoes at moderate levels across geographical regions. We observed that Leptosphaerulina sp. induces a distinctive melanotic phenotype across the developmental stages of mosquito. As a eukaryotic symbiont that is stably associated with An. gambiae Leptosphaerulina sp. can be explored for paratransgenesis.

Abstract Nanotechnology has been a central focus of scientific investigation over the past decades owing to its versatile applications. The synthesis of silver nanoparticles (AgNPs) through plant secondary metabolites is a cost-effective and eco-friendly approach. The present study employed Brassica carinata microgreen extracts (BCME) to promote the reduction of silver nitrate (AgNO3) salt into Brassica carinata microgreen silver nanoparticles (BCM-AgNPs). The physicochemical properties of the biosynthesized AgNPs were characterized through both spectroscopy and microscopy techniques. Furthermore, the antimicrobial property of the biosynthesized AgNPs was assessed against six selected pathogenic microorganisms, and finally, their safety was evaluated on a normal Vero cell line through an MTT cytotoxicity assay. The UV-visible spectrum revealed that BCM-AgNPs exhibited an absorption peak at 420 nm. The potential functional groups involved in the biosynthesis of AgNPs were identified by Fourier transform infrared (FTIR) analysis. Scanning electron microscopy (SEM) revealed a spherical nature of the biosynthesized AgNPs. Transmission electron microscopy (TEM) analysis revealed the crystallinity of the AgNPs, averaging 34.68 nm in size. X-ray diffraction (XRD) investigation further confirmed the crystalline structure of the AgNPs. The zeta potential exhibited a significant value of − 22.5 ± 1.16 mV. Regarding the antimicrobial potential, BCM-AgNPs exhibited promising antimicrobial activity against the tested pathogens, with a minimum inhibitory concentration (MIC) of 62.5 µg/mL observed in Pseudomonas aeruginosa. Further cytotoxicity assessment of BCM-AgNPs conducted on Vero cells demonstrated their safety. This study presents a novel approach to synthesizing AgNPs using a nutraceutical microgreen, offering a biocompatible and promising alternative for combating multi-drug resistance.

Successful optimization of plant-derived compounds into control of nuisance insects would benefit from scientifically validated targets. However, the close association between the genotypic responses and physiological toxicity effects mediated by these compounds remains underexplored. In this study, we evaluated the sublethal dose effects of proanthocyanidins (PAs) sourced from green tea (Camellia sinensis) on life history traits of Anopheles gambiae (sensu stricto) mosquitoes with an aim to unravel the probable molecular targets. Based on the induced phenotypic effects, genes selected for study targeted juvenile hormone (JH) biosynthesis, signal transduction, oxidative stress response and xenobiotic detoxification in addition to vitellogenesis in females. Our findings suggest that chronic exposure of larval stages (L3/L4) to sublethal dose of 5 ppm dramatically extended larval developmental period for up to 12 days, slowed down pupation rates, induced abnormal larval-pupal intermediates and caused 100% inhibition of adult emergence. Further, females exhibited significant interference of fecundity and egg hatchability relative to controls (p < 0.001). Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), our findings show that PA-treated larvae exhibited significant repression of AgamJHAMT (p < 0.001), AgamILP1 (p < 0.001) and AgamCYP6M2 (p < 0.001) with up-regulation of Hsp70 (p < 0.001). Females exposed as larvae demonstrated down-regulation of AgamVg (p = 0.03), AgamILP1 (p = 0.009), AgamCYP6M2 (p = 0.05) and AgamJHAMT (p = 0.02). Our findings support that C. sinensis proanthocyanidins affect important vectorial capacity components such as mosquito survival rates and reproductive fitness thus could be potentially used for controlling populations of malaria vectors.

African trypanosomes thrive in the bloodstream and tissue spaces of a wide range of mammalian hosts. Infections of cattle cause an enormous socio-economic burden in sub-Saharan Africa. A hallmark of the trypanosome lifestyle is the flagellate's incessant motion. This work details the cell motility behavior of the four livestock-parasites Trypanosoma vivax, T. brucei, T. evansi and T. congolense. The trypanosomes feature distinct swimming patterns, speeds and flagellar wave frequencies, although the basic mechanism of flagellar propulsion is conserved, as is shown by extended single flagellar beat analyses. Three-dimensional analyses of the trypanosomes expose a high degree of dynamic pleomorphism, typified by the 'cellular waveform'. This is a product of the flagellar oscillation, the chirality of the flagellum attachment and the stiffness of the trypanosome cell body. The waveforms are characteristic for each trypanosome species and are influenced by changes of the microenvironment, such as differences in viscosity and the presence of confining obstacles. The distinct cellular waveforms may be reflective of the actual anatomical niches the parasites populate within their mammalian host. T. vivax displays waveforms optimally aligned to the topology of the bloodstream, while the two subspecies T. brucei and T. evansi feature distinct cellular waveforms, both additionally adapted to motion in more confined environments such as tissue spaces. T. congolense reveals a small and stiff waveform, which makes these parasites weak swimmers and destined for cell adherence in low flow areas of the circulation. Thus, our experiments show that the differential dissemination and annidation of trypanosomes in their mammalian hosts may depend on the distinct swimming capabilities of the parasites.

Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the Glossina morsitans morsitans (Gmm) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, gmmdripa and gmmdripb ( = gmmaqp1a and gmmaqp1b) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (gmmaqp5, gmmaqp2a, and gmmaqp4b) are expressed highly in the Malpighian tubules. Knockdown of gmmdripa and gmmdripb reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of gmmdripa extended pregnancy length, and gmmdripb knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of gmmdripa, gmmdripb and gmmaqp5 extended pregnancy by 4-6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20-25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.

BackgroundTick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence.MethodsWe screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis.ResultsCandidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study.ConclusionsThe tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Abstract Background The unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity. Methods Malaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico. Results Isoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50 gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50 Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50 IEV = 1.433 µM (95% CI 0.917–2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties. Conclusion These findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

Yam (Dioscorea spp.) is an important food security crop with economic, nutritional, and medicinal value. It is a source of carbohydrates for millions of people in tropical and sub-tropical regions of Africa, Asia, South America, the Caribbean, and the South Pacific Islands. Determining the appropriate parents for breeding programs is the most important decision that plant breeders must make to maximize genetic variability and produce excellent recombinant varieties. However, adequate genetic diversity and the population structure of yam accessions in Kenya are not available to guide accurate selection of parents for breeding. In the present study, 25 start-codon-targeted (SCoT) molecular markers were used to determine the genetic diversity and population structure among 20 yam accessions grown in Kenya. A total of 294 fragments were amplified, of which 95% were polymorphic with an average of 11.16 polymorphic fragments per primer. The polymorphic information content (PIC) value and primer resolving power (Rp) of 0.58 and 5.91, respectively, revealed high genetic diversity among the accessions. A dendrogram based on the unweighted pair group method of arithmetic means (UPGMA) grouped the 20 yam accessions into two clusters at 0.61 genetic similarity coefficients. Bayesian structure analysis revealed the existence of three subpopulations and some admixed accessions. Analysis of molecular variance (AMOVA) revealed a variance of 60% within the subpopulations and 40% among the subpopulations. The high degree of genetic diversity in the yam accessions successfully exhibited by SCoT molecular markers may serve as a valuable aid to widen the genetic base in yam breeding programs. The selection and hybridization of parental lines from the different clusters and sub-clusters identified could provide a foundation and could be exploited for yam breeding and variety development.

Abstract An integrated approach to innovatively counter the transmission of various arthropod-borne diseases to humans would benefit from strategies that sustainably limit onward passage of infective life cycle stages of pathogens and parasites to the insect vectors and vice versa. Aiming to accelerate the impetus towards a disease-free world amid the challenges posed by climate change, discovery, mindful exploitation and integration of active natural products in design of pathogen transmission-blocking interventions is of high priority. Herein, we provide a review of natural compounds endowed with blockade potential against transmissible forms of human pathogens reported in the last 2 decades from 2000 to 2021. Finally, we propose various translational strategies that can exploit these pathogen transmission-blocking natural products into design of novel and sustainable disease control interventions. In summary, tapping these compounds will potentially aid in integrated combat mission to reduce disease transmission trends.

Akagera National Park and its surroundings are home to tsetse flies and a number of their mammalian hosts in Rwanda. A One-health approach is being used in the control and surveillance of both animal and human trypanosomosis in Rwanda. Determination of the infection level in tsetse flies, species of trypanosomes circulating in vectors, the source of tsetse blood meal and endosymbionts is crucial in understanding the epidemiology of the disease in animals and humans in the region.Tsetse flies (n = 1101), comprising Glossina pallidipes (n = 771) and Glossina morsitans centralis (n = 330) were collected from Akagera park and surrounding areas between May 2018 and June 2019. The flies were screened for trypanosomes, vertebrate host DNA to identify sources of blood meal, and endosymbionts by PCR - High Resolution Melting analysis and amplicon sequencing. The feeding frequency and the feeding indices (selection index - W) were calculated to identify the preferred hosts. An overall trypanosome infection rate of 13.9% in the fly's Head and Proboscis (HP) and 24.3% in the Thorax and Abdomen (TA) were found. Eight trypanosome species were identified in the tsetse fly HP and TA, namely: Trypanosoma (T.) brucei brucei, T. congolense Kilifi, T. congolense savannah, T. vivax, T. simiae, T. evansi, T. godfreyi, T. grayi and T. theileri. We found no evidence of human-infective T. brucei rhodesiense. We also identified eighteen species of vertebrate hosts that tsetse flies fed on, and the most frequent one was the buffalo (Syncerus caffer) (36.5%). The frequently detected host by selection index was the rhinoceros (Diceros bicornis) (W = 16.2). Most trypanosome infections in tsetse flies were associated with the buffalo blood meal. The prevalence of tsetse endosymbionts Sodalis and Wolbachia was 2.8% and 4.8%, respectively. No Spiroplasma and Salivary Gland Hypertrophy Virus were detected. These findings implicate the buffaloes as the important reservoirs of tsetse-transmitted trypanosomes in the area. This contributes to predicting the main cryptic reservoirs and therefore guiding the effective control of the disease. The study findings provide the key scientific information that supports the current One Health collaboration in the control and surveillance of tsetse-transmitted trypanosomosis in Rwanda.

Visceral and cutaneous leishmaniasis are endemic to specific regions due to the ecological preferences of phlebotomine sand flies and Leishmania spp. transmission. Sand fly entomological data in northern Kenya are scarce due to limited studies and neglect of leishmaniasis. The aim of this study was to investigate: (i) sand fly diversity and distribution; (ii) occurrence of Leishmania DNA within sand flies; and (iii) blood-meal sources of sand flies in Laisamis, northern Kenya. We conducted an entomological survey during February and March of 2021 in five areas of Laisamis sub-county using standard CDC light traps. A total of 1009 sand flies (394 male and 615 female) were morphologically identified, and representative samples verified by PCR amplification and sequencing of the cytochrome c oxidase subunit 1 (cox1) gene. Similarly, we identified blood-meal sources and Leishmania DNA in female sand flies by PCR amplicon sequencing of the vertebrate cytochrome b (cyt b) gene and internal transcribed spacer 1 (ITS1) of the 28S rRNA gene, respectively. Sergentomyia clydei (59.8%) was the most abundant sand fly species. Though collected mainly from one locality (Tirgamo), 14.8% of samples belonged to Phlebotomus (Artemievus) alexandri Sinton, 1928. We detected DNA of Leishmania major in 5.19% of Ph. alexandri, whereas Leishmania adleri DNA was detected in S. clydei (7.51%), Sergentomyia squamipleuris (8.00%), and Sergentomyia africanus (8.33%). Nine of 13 blood-fed sand flies had obtained blood from humans, of which 33.3% had L. major DNA. Both Ph. alexandri and S. clydei primarily fed on humans and could potentially be involved in the transmission of cutaneous leishmaniasis. The findings of this study contribute to the understanding of sand fly vector populations and their potential to transmit leishmaniasis in the area.

Potato cyst nematodes (PCN), such as Globodera rostochiensis and Globodera pallida, are quarantine restricted pests of potato causing major yield and financial losses to farmers. G. rostochiensis was first reported from Kenya’s key potato growing area in 2015. We sought to determine the diversity, prevalence and distribution of PCN species across the country by conducting a country-wide survey between 2016 and 2018, which included a more focused, follow-up assessment in three key potato growing counties. A total of 1,348 soil samples were collected from 20 potato growing counties. Information regarding local potato farming practices, potato cultivar use, their diversity and availability was also recorded. PCN cysts were obtained from 968 samples (71.8%) in all the counties surveyed, with Nyandarua County recording the highest PCN field-incidence at 47.6%. The majority of PCN populations, 99.9%, were identified as G. rostochiensis, while G. pallida was recovered from just one field, in a mixed population with G. rostochiensis. Inconsistencies in PCR amplification efficiency was observed for G. rostochiensis using the recommended EPPO primers, compared with ITS primers AB28/TW81, indicating that this protocol cannot be entirely relied upon to effectively detect PCN. Egg density in Nyandarua County varied between 30.6 and 158.5 viable eggs/g soil, with an average egg viability of 78.9 ± 2.8% (min = 11.6%, max = 99.9%). The PCN-susceptible potato cultivar named Shangi was the most preferred and used by 65% of farmers due to its shorter dormancy and cooking time, while imported cultivars (Destiny, Jelly, Manitou, and Markies) with resistance to G. rostochiensis were used by 7.5% of farmers due to unavailability and/or limited access to seeds. Thus, most farmers preferred using their own farm-saved seeds as opposed to purchasing certified seeds. Establishing the distribution and prevalence of PCN and elucidating the local farming practices that could promote the spread of PCN is a necessary precursor to the implementation of any containment or management strategy in the country and ultimately across the region.

Abstract Globally, most gut microbiota-related studies have focused on broilers due to their diverse microbial communities compared to that of layer chicken. However, in Africa few studies have been undertaken despite the increasing benefits to the poultry industry. The utilization of Insect-Based diets to improve the gastrointestinal function and gut health in poultry is increasingly gaining global attention. Here, we evaluated the potential roles of commercial black soldier fly larvae-based feeds (BSFLF) in reshaping the abundance, composition and diversity of the gut microbiota of layer chickens using high throughput Oxford nanopore Minion sequencing of the full length bacterial 16S rRNA gene. Two hundred and fifty ISA Brown layer chicks were reared in pens for a period of 20 weeks. The layer pullets were allotted 5 dietary treatments that were formulated as follows: control diet (T1): 100% FM + 0% BSFL, T2: 25% BSFL + 75% FM; T3: 50% BSFL + 50% FM; T4: 75% BSFL + 25% FM, and T5: 100% BSFL + 0% FM. Sampling was done from the eight major regions including oesophagus, crop, proventriculus, gizzard, duodenum, ileum, large intestines and ceca. Out of the 400,064 classified reads analyzed, the most dominant phyla identified across the feed treatments were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. The diet treatment with 100% inclusion levels of BSFL showed the highest intra-species alpha diversity and richness according to Chao1 and Shannon index. Intra-species beta diversity assessment revealed that the diet types significantly influenced the abundance of the microbiota, but differences between most abundant taxa were similar. There was increase in abundance of potentially beneficial bacteria (Lactobacillus, Bacteroides and Enterococcus) with increased inclusion levels of BSFLF in layer pullets diets. Across the different gut segments, Lactobacillus dominated all the eight regions and the ceca was the most diverse segment. Our findings unravel complex gut microbial shift in laying hen fed BSFLF and therefore underpins the potential roles of beneficial bacteria as promising prebiotics and probiotics in reshaping of the gut microbiota to maintain good gut health.

ABSTRACT Drug-resistant strains of Pseudomonas aeruginosa and Candida albicans pose serious threats to human health because of their propensity to cause fatal infections. Defensin and defensin-like antimicrobial peptides (AMPs) are being explored as new lines of antimicrobials, due to their broad range of activity, low toxicity, and low pathogen resistance. Defensin-d2 and actifensin are AMPs from spinach and Actinomyces ruminicola, respectively, whose mechanisms of action are yet to be clearly elucidated. This study investigated the mechanisms of action of the recombinant AMPs through label-free quantitative proteomics. The data are available at PRIDE with accession number PXD034169. A total of 28 and 9 differentially expressed proteins (DEPs) were identified in the treated P. aeruginosa and C. albicans, respectively, with a 2-fold change threshold and P values of

There is an increased demand for natural products like propolis, yet little information is available about the chemical composition of African propolis and its bio-functional properties. Therefore, in this study, we aimed to quantify the phytochemicals and determine the antioxidant and antimicrobial properties of Apis mellifera propolis (n = 59) sourced from various regions in Kenya. Principal component analysis (PCA) showed that the sampling region had a remarkable impact on the propolis's composition and bio-functional properties. Generally, the propolis contained high amounts of phytochemicals, particularly alkaloids (5.76 g CE/100 g) and phenols (2.24 g GAE/100 g). Furthermore, analysis of propolis by gas chromatography–mass spectrometry (GC-MS) revealed various compounds with varying bio-functional activities. These compounds included triterpenoids alpha- and beta-amyrin, oleanen-3-yl-acetate, urs-12-en-24-oic acid, lanosta-8,24-dien-3-one, and hydrocarbons tricosane and nondecane, which have been reported to have either antimicrobial or antioxidant activities. The propolis samples collected from hotter climatic conditions contained a higher composition of phytochemicals, and additionally, they displayed higher antioxidant and antimicrobial activities than those obtained from cooler climatic conditions. Key findings of this study demonstrate the occurrence of relatively high phytochemical content in Kenya's propolis, which has antioxidant and antimicrobial properties; hence this potential could be harnessed for disease control.

Abstract Background Plasmodium falciparum parasite populations in Ethiopia have been experiencing local selective pressures from drugs and immunity, leading to evolutionary adaptation. However, there was a paucity of data on genomic characterization and evolutionary adaptations of P. falciparum isolates from the central area of Ethiopia. Methods Whole-genome analysis of 25 P. falciparum isolates from central Ethiopia, specifically from West Arsi, were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles against global isolates from Cambodia, Thailand, DR Congo, and Malawi. Results A total of 18,517 high-quality single-nucleotide polymorphisms (SNPs) were identified in Ethiopian P. falciparum isolates. About 84% of the Ethiopian P. falciparum isolates had a FWS value > 0.95 showing a dominant single genotype infection in most isolates at the time of collection with little potential for out-crossing as expected in areas with low transmission intensity. Within-host diversity of Ethiopian infections was significantly different from East African (p 0.05). A significant population structure has been observed by PCA and population differentiation between Ethiopian parasites and East African (Fst ~ 10%) and Southeast Asian populations (Fst ~ 18%), suggesting limited gene flow and the independent evolution of the Ethiopian parasite population. Moreover, a total of 125 genes under balancing selection was found that include ama1, trap, eba175, and lsa3, previously identified as targets of human host immunity. Recent directional selection analysis using integrated standardized haplotype score (IHS) did not detect any selection signatures in the Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. However, known drug resistance-conferring mutations analysis showed that at least one SNP marker was fixed in these genes, but not in Pfdhps and PfK13. Conclusion Plasmodium falciparum populations in the central region of Ethiopia was structurally diverged from both Southeast Asian and other East African populations. Malaria infections in Ethiopia had low within-host diversity, and parasites carry fixed chloroquine resistance markers despite the withdrawal of this drug for the treatment of P. falciparum.

Abstract Background Trypanosoma brucei species are motile protozoan parasites that are cyclically transmitted by tsetse fly (genus Glossina) causing human sleeping sickness and nagana in livestock in sub-Saharan Africa. African trypanosomes display digenetic life cycle stages in the tsetse fly vector and in their mammalian host. Experimental work on insect-stage trypanosomes is challenging because of the difficulty in setting up successful in vitro cultures. Therefore, there is limited knowledge on the trypanosome biology during its development in the tsetse fly. Consequently, this limits the development of new strategies for blocking parasite transmission in the tsetse fly. Methods In this study, RNA-Seq data of insect-stage trypanosomes were used to construct a T. brucei gene co-expression network using the weighted gene co-expression analysis (WGCNA) method. The study identified significant enriched modules for genes that play key roles during the parasite’s development in tsetse fly. Furthermore, potential 3′ untranslated region (UTR) regulatory elements for genes that clustered in the same module were identified using the Finding Informative Regulatory Elements (FIRE) tool. Results A fraction of gene modules (12 out of 27 modules) in the constructed network were found to be enriched in functional roles associated with the cell division, protein biosynthesis, mitochondrion, and cell surface. Additionally, 12 hub genes encoding proteins such as RNA-binding protein 6 (RBP6), arginine kinase 1 (AK1), brucei alanine-rich protein (BARP), among others, were identified for the 12 significantly enriched gene modules. In addition, the potential regulatory elements located in the 3′ untranslated regions of genes within the same module were predicted. Conclusions The constructed gene co-expression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. This will enhance understanding of the molecular mechanisms that underlie important biological processes during parasite’s development in tsetse fly. Ultimately, these findings will be key in the identification of potential molecular targets for disease control.

Background. Tsetse flies are vectors of the genus Trypanosoma that cause African trypanosomiasis, a serious parasitic disease of people and animals. Reliable data on the vector distribution and the trypanosome species they carry is pertinent for planning sustainable control strategies. This study was carried out to estimate the spatial distribution, apparent density, and trypanosome infection rates of tsetse flies in two districts that fall within a vector genetic transition zone in northern Uganda. Materials and Methods. Capturing of tsetse flies was done using biconical traps deployed in eight villages in Oyam and Otuke, two districts that fall within the vector genetic transition zone in northern Uganda. Trapped tsetse flies were sexed and morphologically identified to species level and subsequently analyzed for detection of trypanosome DNA. Trypanosome DNA was detected using a nested PCR protocol based on primers amplifying the internal transcribed spacer (ITS) region of ribosomal DNA. Results. A total of 717 flies (406 females; 311 males) were caught, all belonging to the Glossina fuscipes fuscipes species. The overall average flies/trap/day (FTD) was 2.20±0.3527 (mean±SE). Out of the 477 (201 male; 276 females) flies analyzed, 7.13% (34/477) were positive for one or more trypanosome species. Three species of bovine trypanosomes were detected, namely, Trypanosoma vivax, 61.76% (21/34), T. congolense, 26.47% (9/34), and T. brucei brucei, 5.88% (2/34), and two cases of mixed infection of T. congolense and T. brucei brucei, 5.88% (2/34). The infection rate was not significantly associated with the sex of the fly (generalized linear model (GLM), χ2=0.051, p=0.821, df=1,n=477) and district of origin (χ2=0.611, p=0.434, df=1, n=477). However, trypanosome infection was highly significantly associated with the fly’s age based on wing fray category (χ2=7.56, p=0.006, df=1, n=477), being higher among the very old than the young. Conclusion. The relatively high tsetse density and trypanosome infection rate indicate that the transition zone is a high-risk area for perpetuating animal trypanosomiasis. Therefore, appropriate mitigation measures should be instituted targeting tsetse and other biting flies that may play a role as disease vectors, given the predominance of T. vivax in the tsetse samples.

The recently discovered Anopheles symbiont, Microsporidia MB, has a strong malaria transmission-blocking phenotype in Anopheles arabiensis, the predominant Anopheles gambiae species complex member in many active transmission areas in eastern Africa. The ability of Microsporidia MB to block Plasmodium transmission together with vertical transmission and avirulence makes it a candidate for the development of a symbiont-based malaria transmission blocking strategy. We investigate the characteristics and efficiencies of Microsporidia MB transmission between An. arabiensis mosquitoes. We show that Microsporidia MB is not transmitted between larvae but is effectively transmitted horizontally between adult mosquitoes. Notably, Microsporidia MB was only found to be transmitted between male and female An. arabiensis, suggesting sexual horizontal transmission. In addition, Microsporidia MB cells were observed infecting the An. arabiensis ejaculatory duct. Female An. arabiensis that acquire Microsporidia MB horizontally are able to transmit the symbiont vertically to their offspring. We also investigate the possibility that Microsporidia MB can infect alternate hosts that live in the same habitats as their An. arabiensis hosts, but find no other non-anopheline hosts. Notably, Microsporidia MB infections were found in another primary malaria African vector, Anopheles funestus s.s. The finding that Microsporidia MB can be transmitted horizontally is relevant for the development of dissemination strategies to control malaria that are based on the targeted release of Microsporidia MB infected Anopheles mosquitoes.

The utilization of insect-based diets to improve gastrointestinal function and gut health in poultry is gaining global attention as a promising feed additive. The objective of this study was to determine the optimal inclusion level of the full-fat black soldier fly larvae (BSFL) and Desmodium intortum (DI) in broiler chicken diets and to evaluate their impact on the microbial community in the gut. The bacterial communities were characterized using Oxford nanopore sequencing of the full-length bacterial 16S rRNA gene. Four dietary treatments, T1 (25% DI + 75% BSFL), T2 (50% DI + 50% BSFL), T3 (75% DI + 25% BSFL) and T4 (100% fishmeal + 0% DI + BSFL), were fed to the broiler chickens for a period of 42 days. Out of the 395,034 classified reads analyzed, the most predominant phyla identified across all the four dietary treatments were Firmicutes (94%), Bacteroidetes (3%), and Proteobacteria (2%). The T1 diet showed the highest alpha diversity and richness according to the Chao1 and Shannon indices. Beta diversity assessment revealed a significant influence of diet on the abundance of the microbiome. There was an increase in beneficial lactic acid bacteria with increasing inclusion of BSFL in the diets. Our findings strongly support the inclusion of BSFL into poultry diet as a promising protein source to reshape the gut microbiota for improved gut health, immune response, and food safety.

Antimicrobial resistance requires urgent efforts towards the discovery of active antimicrobials, and the development of strategies to sustainably produce them. Defensin and defensin-like antimicrobial peptides (AMPs) are increasingly gaining pharmacological interest because of their potency against pathogens. In this study, we expressed two AMPs: defensin-d2 derived from spinach, and defensin-like actifensin from Actinomyces ruminicola. Recombinant pTXB1 plasmids carrying the target genes encoding defensin-d2 and actifensin were generated by the MEGAWHOP cloning strategy. Each AMP was first expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and was thereafter assayed for antimicrobial activity against multidrug-resistant (MDR) pathogens. Approximately 985 µg/mL and 2895 µg/mL of recombinant defensin-d2 and actifensin, respectively, were recovered with high purity. An analysis by MALDI-TOF MS showed distinct peaks corresponding to molecular weights of approximately 4.1 kDa for actifensin and 5.8 kDa for defensin-d2. An in vitro antimicrobial assay showed that MDR Pseudomonas aeruginosa and Candida albicans were inhibited at minimum concentrations of 7.5 µg/mL and 23 µg/mL for recombinant defensin-d2 and actifensin, respectively. The inhibitory kinetics of the peptides revealed cidal activity within 4 h of the contact time. Furthermore, both peptides exhibited an antagonistic interaction, which could be attributed to their affinities for similar ligands, as deduced by peptide–ligand profiling. Moreover, both peptides inhibited biofilm formation, and they exhibited no resistance potential and low hemolytic activity. The peptides also possess the ability to permeate and disrupt the cell membranes of MDR P. aeruginosa and C. albicans. Therefore, recombinant actifensin and defensin-d2 exhibit broad-spectrum antimicrobial activity and have the potential to be used as therapy against MDR pathogens.

Stomoxys calcitrans (stable fly) is a cosmopolitan biting fly of both medical and veterinary importance. Unlike blood-feeding-related behavior of stable fly, its plant feeding, the fitness value, and the S. calcitrans–plant interaction are less understood. Here we show based on two chloroplast DNA genes, ribulose bisphosphate carboxylase large chain (rbcL) and the intergenic spacer gene trnH-psbA, that field-collected male and female stable flies fed on various plant species. We investigated the fitness cost of plant feeding using Parthenium hysterophorus, one of the plant species identified to have been fed on by the field-collected flies. Supplementation of blood feeding with a flowering P. hysterophorus plant as nectar source enhanced egg hatchability significantly as compared to blood alone, showing the fitness value of nectar supplementation. However, nectar supplementation did not affect the number of eggs laid or longevity of S. calcitrans as compared to flies that fed on blood alone. S. calcitrans maintained on sugar alone failed to lay eggs. The various plants stable flies fed on demonstrated chemodiversity with their own signature scent. The behavioral response of S. calcitrans to these signature compounds varied from strong attraction (γ-terpinene) to neutral (linalool oxide and myrcene) to repellency (butanoic acid). Our study demonstrated that stable flies feed on nectar, and plant nectar supplementation of blood feeding enhanced larval emergence. Thus, our result has implication in stable fly reproduction, survival, disease transmission, boosting laboratory colony, and the possibility of using plant-derived odors for mass trapping of stable fly, for instance, using γ-terpinene.

A disease with clinical and post-mortem presentation similar to those seen in heartwater, a tick-borne disease of domestic and wild ruminants caused by the intracellular bacterium Ehrlichia ruminantium, was first reported in dromedary camels in Kenya in 2016; investigations carried out at the time to determine the cause were inconclusive. In the present study, we screened sera from Kenyan camels collected before (2015) and after (2020) the 2016 disease outbreak for antibodies to Ehrlichia spp. using an E. ruminantium polyclonal competitive ELISA (PC-ELISA). Median antibody levels were significantly higher (p < 0.0001) amongst camels originating from areas where the heartwater-like disease was reported than from disease-free areas, for animals sampled in both 2015 and 2020. Overall median seropositivity was higher in camels sampled in 2015 than in 2020, which could have been due to higher mean age in the former group. Camels that were PCR-positive for Candidatus Ehrlichia regneryi had significantly lower (p = 0.03) median antibody levels than PCR-negative camels. Our results indicate that Kenyan camels are frequently exposed to E. ruminantium from an early age, E. ruminantium was unlikely to have been the sole cause of the outbreak of heartwater-like disease; and Ca. E. regneryi does not appreciably cross-react with E. ruminantium in the PC-ELISA.

Background: Major constraints to camel production include pests and diseases. In northern Kenya, little information is available about blood-borne pathogens circulating in one-humped camels ( Camelus dromedarius) or their possible transmission by the camel haematophagous ectoparasite, Hippobosca camelina, commonly known as camel ked or camel fly. This study aimed to: (i) identify the presence of potentially insect-vectored pathogens in camels and camel keds, and (ii) assess the potential utility of keds for xenodiagnosis of camel pathogens that they may not vector. Methods: In Laisamis, northern Kenya, camel blood samples (n = 249) and camel keds (n = 117) were randomly collected from camels. All samples were screened for trypanosomal and camelpox DNA by PCR, and for Anaplasma, Ehrlichia, Brucella, Coxiella, Theileria, and Babesia by PCR coupled with high-resolution melting (PCR-HRM) analysis. Results: In camels, we detected Trypanosoma vivax (41%), Trypanosoma evansi (1.2%), and “ Candidatus Anaplasma camelii” (68.67%). In camel keds, we also detected T. vivax (45.3%), T. evansi (2.56%), Trypanosoma melophagium (1/117) (0.4%), and “ Candidatus Anaplasma camelii” (16.24 %). Piroplasms ( Theileria spp. and Babesia spp.), Coxiella burnetii, Brucella spp., Ehrlichia spp., and camel pox were not detected in any samples. Conclusions: This study reveals the presence of epizootic pathogens in camels from northern Kenya. Furthermore, the presence of the same pathogens in camels and in keds collected from sampled camels suggests the potential use of these flies in xenodiagnosis of haemopathogens circulating in camels.

Abstract Malaria presents an overwhelming public health challenge, particularly in sub-Saharan Africa where vector favourable conditions and poverty prevail, potentiating the disease burden. Behavioural variability of malaria vectors poses a great challenge to existing vector control programmes with insecticide resistance already acquired to nearly all available chemical compounds. Thus, approaches incorporating plant-derived compounds to manipulate semiochemical-mediated behaviours through disruption of mosquito olfactory sensory system have considerably gained interests to interrupt malaria transmission cycle. The combination of push-pull methods and larval control have the potential to reduce malaria vector populations, thus minimising the risk of contracting malaria especially in resource-constrained communities where access to synthetic insecticides is a challenge. In this review, we have compiled information regarding the current status of knowledge on manipulation of larval ecology and chemical-mediated behaviour of adult mosquitoes with plant-derived compounds for controlling mosquito populations. Further, an update on the current advancements in technologies to improve longevity and efficiency of these compounds for field applications has been provided.

Ticks and tick-borne pathogens (TBPs) are major constraints to camel health and production, yet epidemiological data on their diversity and impact on dromedary camels remain limited. We surveyed the diversity of ticks and TBPs associated with camels and co-grazing sheep at 12 sites in Marsabit County, northern Kenya. We screened blood and ticks (858 pools) from 296 camels and 77 sheep for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products. Hyalomma (75.7%), Amblyomma (17.6%) and Rhipicephalus (6.7%) spp. ticks were morphologically identified and confirmed by molecular analyses. We detected TBP DNA in 80.1% of blood samples from 296 healthy camels. “Candidatus Anaplasma camelii”, “Candidatus Ehrlichia regneryi” and Coxiella burnetii were detected in both camels and associated ticks, and Ehrlichia chaffeensis, Rickettsia africae, Rickettsia aeschlimannii and Coxiella endosymbionts were detected in camel ticks. We also detected Ehrlichia ruminantium, which is responsible for heartwater disease in ruminants, in Amblyomma ticks infesting camels and sheep and in sheep blood, indicating its endemicity in Marsabit. Our findings also suggest that camels and/or the ticks infesting them are disease reservoirs of zoonotic Q fever (C. burnetii), ehrlichiosis (E. chaffeensis) and rickettsiosis (R. africae), which pose public health threats to pastoralist communities.

Tick-borne pathogens (TBPs) are major constraints to livestock production and a threat to public health in Africa. This cross-sectional study investigated the risk of infection with TBPs in cattle of Lambwe Valley, Kenya. Blood samples of 680 zebu cattle from 95 herds in six geospatial clusters within 5 km of Ruma National Park were screened for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products. We detected Anaplasma bovis (17.4%), Anaplasma platys (16.9%), Anaplasma marginale (0.6%), Theileria velifera (40%), and Theileria mutans (25.7%), as well as an Anaplasma sp. (11.6%) that matched recently reported Anaplasma sp. sequences from Ethiopia. Babesia, Rickettsia, and Ehrlichia spp. were not detected. The animal and herd-level prevalences for TBPs were 78.5% (95% confidence intervals (CI): 75.3, 81.5) and 95.8% (95% CI: 91.8, 99.8), respectively. About 31.6% of cattle were co-infected with 13 combinations of TBPs. The prevalence of TBPs differed between clusters and age, but the risk of infection was not associated with sex, herd size, or the distance of homesteads from Ruma. This study adds insight into the epidemiology of TBPs around Ruma and highlights the need for proactive surveillance of TBPs in livestock–wildlife interfaces.