Attractant and repellent properties of Senna didymobotrya plant extracts to Amblyomma variegatum and Rhipicephalus appendiculatus.

The growing challenge of acaricide resistance and geographical range expansion of invasive tick species demands other interventions, like plant-based alternatives, for sustainable tick control. Leaves, flowers, seedpods, and twig branch extracts of Senna didymobotrya were analyzed using coupled gas chromatography mass spectrometry (GC-MS). Response of adult Amblyomma variegatum and Rhipicephalus appendiculatus to extracts was evaluated. The most attractive plant extract was fractionated and ticks' responses to its fractions assessed. Potential tick attractants in the attractive plant part extract and its fractions were identified by GC-MS analysis. Non- significant qualitative and quantitative differences were observed in the plant parts' extract composition (R = 0.6178). Flower extracts attracted both species, with a 0.1-fold higher attraction in A. variegatum compared to the standard attraction aggregation attachment pheromone (AAAP). Leaf and seedpod extracts repelled ticks at various concentrations. Bioassays after fractionating flower extracts identified hexane and ethyl acetate fractions as most attractive to A. variegatum (P < 0.001) and R. appendiculatus (P < 0.001), respectively. Chemical analysis of the most attractive extracts and fractions identified compounds, including documented acarine attractants, squalene and linoleic acid. A squalene and linoleic acid blend (1:1) at 1 mg/mL significantly attracted adult A. variegatum (P < 0.01) and R. appendiculatus (P < 0.001). The results of this study broaden comprehension of how ticks respond to plants in nature, and showcase the promising potential for integrating these insights into effective tick management programs.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-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. Methods: We 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. Results: Candidatus 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. Conclusions: The 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.

A scoping review on tsetse fly blood meal sources and its assay methods since 1956 to 2022.

BACKGROUND: Tsetse flies (Glossina spp.) are the definitive biological vectors of African trypanosomes in humans and animals. Controlling this vector is the most promising method of preventing trypanosome transmission. This requires a comprehensive understanding of tsetse biology and host preference to inform targeted design and management strategies, such as the use of olfaction and visual cues in tsetse traps. No current review exists on host preference and blood meal analyses of tsetse flies. METHODS: This review presents a meta-analysis of tsetse fly blood meal sources and the methodologies used to identify animal hosts from 1956 to August 2022. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRIMA-ScR) was applied. This focused on tsetse-endemic countries, blood meal analysis methodologies and the blood meal hosts identified. The articles were retrieved and screened from databases using predetermined eligibility criteria. RESULTS: Only 49/393 of the articles retrieved matched the inclusion criteria. Glossina's main hosts in the wild included the bushbuck, buffalo, elephant, warthog, bushpig and hippopotamus. Pigs, livestock and humans were key hosts at the domestic interface. The least studied species included Glossina fuscipleuris, G. fusca, G. medicorum, G. tabaniformis and G. austeni. In the absence of preferred hosts, Glossina fed opportunistically on a variety of hosts. Precipitin, haemagglutination, disc diffusion, complement fixation, ELISA and PCR-based assays were used to evaluate blood meals. Cytochrome b (Cyt b) was the main target gene in PCR to identify the vertebrate hosts. CONCLUSIONS: Tsetse blood meal sources have likely expanded because of ecological changes that could have rendered preferred hosts unavailable. The major approaches for analysing tsetse fly blood meal hosts targeted Cyt b gene for species identification by Sanger sequencing. However, small-fragment DNAs, such as the mammalian 12S and 16S rRNA genes, along with second- and third-generation sequencing techniques, could increase sensitivity for host identification in multiple host feeders that Sanger sequencing may misidentify as "noise". This review of tsetse fly blood meal sources and approaches to host identification could inform strategies for tsetse control.

Discovery of the vector of visceral leishmaniasis, Phlebotomus (Artemievus) alexandri Sinton, 1928, in Kenya suggests complex transmission dynamics.

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.

Decadal Assessment of Microplastics, Pharmaceuticals, and Pesticides as Contaminants of Emerging Concern in Kenya's Surface Waters: A Review.

Studies investigating microplastics, pharmaceuticals, and pesticides as contaminants of emerging concern (CECs) in surface water sources in Kenya are reviewed. Contaminants of emerging concern are chemicals that have recently been discovered that may pose a threat to the environment, aquatic life, and human life. Microplastics in surface waters range from 1.56 to as high as 4520 particles/m3 , with high concentrations recorded in coastal waters. The dominant microplastics are fibers, fragments, and films, with foams, granules, and pellets making up only a small percentage. The main source of pharmaceuticals in water sources is not wastewater-treatment plants but rather raw untreated sewage because high concentrations are found near informal settlements with poor sewage connectivity. Antibiotics are detected in the range of the limit of quantification to 320 µg/L, with sulfamethoxazole, trimethoprim, and ciprofloxacin being the most abundant antibiotics. The high frequency of detection is attributed to the general misuse of antibiotics in the country. A health risk assessment indicated that only ciprofloxacin and acetaminophen posed noncarcinogenic health risks in the Ndarugo River and Mombasa periurban creeks, respectively. Similarly, the detection of antiretroviral drugs, mainly lamivudine, nevirapine, and zidovudine, is associated with human immunodeficiency virus prevalence in Kenya. In the Lake Naivasha, Nairobi River, and Lake Victoria basins, frequently detected organochloride pesticides (OCPs) are methoxychlor, alachlor, endrin, dieldrin, endosulfan, endosulfan sulfate, α-hexachlorocyclohexane (α-HCH), γ-HCH, and dichlorodiphenyltrichloroethane (DDT), some of which occur above permissible concentrations. The presence of DDT in some sites translates to illegal use or historical application. The majority of individual OCPs posed no noncarcinogenic health risk, except dieldrin and aldrin which had a hazard quotient >1 in two sites. Therefore, more surveying and regular monitoring in different regions in Kenya concerning CECs is essential to determine the spatial variability and effective measures to be taken to reduce pollution. Environ Toxicol Chem 2023;42:2105-2118. © 2023 SETAC.

Trypanosomes infection, endosymbionts, and host preferences in tsetse flies (Glossina spp.) collected from Akagera park region, Rwanda: A correlational xenomonitoring study.

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.

Genomic epidemiology of the rotavirus G2P[4] strains in coastal Kenya pre- and post-rotavirus vaccine introduction, 2012-8.

The introduction of rotavirus vaccines into the national immunization programme in many countries has led to a decline in childhood diarrhoea disease burden. Coincidentally, the incidence of some rotavirus group A (RVA) genotypes has increased, which may result from non-vaccine-type replacement. Here, we investigate the evolutionary genomics of rotavirus G2P[4] which has shown an increase in countries that introduced the monovalent Rotarix® vaccine. We examined sixty-three RVA G2P[4] strains sampled from children (aged below 13 years) admitted to Kilifi County Hospital, coastal Kenya, pre- (2012 to June 2014) and post-(July 2014 to 2018) rotavirus vaccine introduction. All the sixty-three genome sequences showed a typical DS-1-like genome constellation (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Pre-vaccine G2 sequences predominantly classified as sub-lineage IVa-3 and co-circulated with low numbers of sub-lineage IVa-1 strains, whereas post-vaccine G2 sequences mainly classified into sub-lineage IVa-3. In addition, in the pre-vaccine period, P[4] sub-lineage IVa strains co-circulated with low numbers of P[4] lineage II strains, but P[4] sub-lineage IVa strains predominated in the post-vaccine period. On the global phylogeny, the Kenyan pre- and post-vaccine G2P[4] strains clustered separately, suggesting that different virus populations circulated in the two periods. However, the strains from both periods exhibited conserved amino acid changes in the known antigenic epitopes, suggesting that replacement of the predominant G2P[4] cluster was unlikely a result of immune escape. Our findings demonstrate that the pre- and post-vaccine G2P[4] strains circulating in Kilifi, coastal Kenya, differed genetically but likely were antigenically similar. This information informs the discussion on the consequences of rotavirus vaccination on rotavirus diversity.

Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates.

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 IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual 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 IC50IEV = 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.

Oral acute, sub-acute toxicity and phytochemical profile of Brassica carinata A. Braun microgreens ethanolic extract in Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Currently, there is a remarkable increase in the consumption of microgreens, (young edible vegetables or herbs), as potential nutraceuticals for the management of diseases. Brassica carinata A. Braun is one of the traditional leafy vegetables cultivated in various parts of Sub- Saharan Africa. The plant is revered for its efficacy in the treatment of wounds and gastrointestinal disorders among other medicinal benefits. It is therefore crucial to characterize Brassica carinata microgreens for their phytoconstituents and ascertain their safety for use. AIM OF THE STUDY: The study evaluated the oral acute and subacute toxicity of Brassica carinata microgreens ethanol extract (BMEE) in Wistar rats and identification of its chemical composition and profile. MATERIALS AND METHODS: For acute toxicity (14 days), rats were grouped into four and received a single oral dose, the control group received distilled water, while others received 500 mg/kg, 1000 mg/kg, and 2000 mg/kg of BMEE. For the subacute toxicity (28 days), rats in four groups received daily doses of 250 mg/kg, 500 mg/kg or 1000 mg/kg and distilled water. Daily clinical observations like lethargy and mortality were conducted. Hematological, biochemical, and histopathological evaluations were performed at the end of each experiment. Phytochemical profile was determined using a UV-VIS spectrophotometer and Gas Chromatography coupled to Mass Spectrometry (GC-MS) analysis determined the potential bioactive components in the microgreens extract. RESULTS: In both acute and sub-acute toxicity studies, no mortalities, indications of abnormality, or any treatment related adverse effects were observed at doses of 2000 mg/kg, 1000 mg/kg, 500 mg/kg, and 250 mg/kg. The LD50 of BMEE was above 2000 mg/kg. No significant (p > 0.05) changes in the hematological and biochemical parameters of the treated groups compared to the control groups in both studies. Histopathological examination of the liver, kidney, lungs, and heart revealed a normal architecture of the tissues in all the treated animals. Phytochemical analyses revealed the presence of flavonoids (most abundant), phenols and alkaloids. Phytol, linoleic acid, and 9,12,15-octadecatrienoic acid, among other compounds, were identified by GC-MS analysis. CONCLUSION: The results showed that B. carinata microgreens ethanol extract is nontoxic and found to have several compounds with reported pharmacological significance suggesting safety for use.

Gut microbiota shift in layer pullets fed on black soldier fly larvae-based feeds towards enhancing healthy gut microbial community.

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.

Recombinant Actifensin and Defensin-d2 Induce Critical Changes in the Proteomes of Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans.

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 <0.05. Functional analysis revealed that the DEPs were involved in DNA replication and repair, translation, and membrane transport in P. aeruginosa, while they were related mainly to oxidative phosphorylation, RNA degradation, and energy metabolism in C. albicans. Protein-protein interactions showed that the DEPs formed linear or interdependent complexes with one another, indicative of functional interaction. Subcellular localization indicated that the majority of DEPs were cytoplasmic proteins in P. aeruginosa, while they were of nuclear or mitochondrial origin in C. albicans. These results show that recombinant defensin-d2 and actifensin can elicit complex multiple organism responses that cause cell death in P. aeruginosa and C. albicans. IMPORTANCE AMPs are considered essential alternatives to conventional antimicrobials because of their broad-spectrum efficacy and low potential for resistance by target cells. In this study, we established that the recombinant AMPs defensin-d2 and actifensin exert proteomic changes in P. aeruginosa and C. albicans within 1 h after treatment. We also found that the DEPs in peptide-treated P. aeruginosa are related to ion transport and homeostasis, molecular functions including nucleic and amino acid metabolism, and structural biogenesis and activity, while the DEPs in treated C. albicans are mainly involved in membrane synthesis and mitochondrial metabolism. Our results also highlight ATP synthase as a potential drug target for multidrug-resistant P. aeruginosa and C. albicans.

Phytochemical composition and bio-functional properties of Apis mellifera propolis from Kenya.

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.

Contemporary exploitation of natural products for arthropod-borne pathogen transmission-blocking interventions.

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.

Insights into the Gut Microbial Communities of Broiler Chicken Fed Black Soldier Fly Larvae-Desmodium-Based Meal as a Dietary Protein Source.

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.

Membrane Permeabilization and Antimicrobial Activity of Recombinant Defensin-d2 and Actifensin against Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans.

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.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Spatial Distribution of Tsetse Flies and Trypanosome Infection Status in a Vector Genetic Transition Zone in Northern Uganda.

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), χ 2 = 0.051, p = 0.821, df = 1, n = 477) and district of origin (χ 2 = 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 (χ 2 = 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.

Detection of Antibodies to Ehrlichia spp. in Dromedary Camels and Co-Grazing Sheep in Northern Kenya Using an Ehrlichia ruminantium Polyclonal Competitive ELISA.

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.

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.

Farmers' knowledge, perceptions, and practices on animal trypanosomosis and the tsetse fly vector: A cross-sectional study around Kenya's Arabuko-Sokoke Forest Reserve at the livestock-wildlife interface.

Background: Animal African trypanosomosis (AAT) is a veterinary disease caused by trypanosomes transmitted cyclically by tsetse flies. AAT causes huge agricultural losses in sub-Saharan Africa. Both tsetse flies and trypanosomosis (T&T) are endemic in the study area inhabited by smallholder livestock farmers at the livestock-wildlife interface around Arabuko-Sokoke Forest Reserve (ASFR) in Kilifi County on the Kenyan coast. We assessed farmers' knowledge, perceptions and control practices towards T&T. Methods: A cross-sectional study was conducted during November and December 2017 to collect data from 404 randomly selected cattle-rearing households using a structured questionnaire. Descriptive statistics were used to determine farmers' knowledge, perceptions, and control practices towards T&T. Demographic factors associated with knowledge of T&T were assessed using a logistic regression model. Results: Participants consisted of 53% female, 77% married, 30% elderly (>55 years), and the majority (81%) had attained primary education or below. Most small-scale farmers (98%) knew the tsetse fly by its local name, and 76% could describe the morphology of the adult tsetse fly by size in comparison to the housefly's ( Musca domestica). Only 16% of the farmers knew tsetse flies as vectors of livestock diseases. Higher chances of adequate knowledge on T&T were associated with the participants' (i) age of 15-24 years (aOR 2.88 (95% CI 1.10-7.52), (ii) level of education including secondary (aOR 2.46 (95% CI 1.43-4.24)) and tertiary (aOR 3.80 (95% CI 1.54-9.37)), and (iii) employment status: self-employed farmers (aOR 6.54 (95% CI 4.36-9.80)). Conclusions: Our findings suggest that small-scale farmers around ASFR have limited knowledge of T&T. It is envisaged that efforts geared towards training of the farmers would bridge this knowledge gap and sharpen the perceptions and disease control tactics to contribute to the prevention and control of T&T.