Mabel Imbuga

Molecular Characterization of the Cytochrome b Gene and In Vitro Atovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya

ABSTRACT The prevalence of a genetic polymorphism(s) at codon 268 in the cytochrome b gene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227 Plasmodium falciparum parasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

Comparison of Midgut Trypsin/Lectin Activities and Trypanosome Infection Rates in Three Glossina Species

Midgut trypsin and lectin levels were determined in three tsetse species, namely Glossina morsitans morsitans, G. longipennis and G. fuscipes fuscipes. In addition, the abilities of midgut homogenates prepared from these flies to transform bloodstream-form Trypanosoma brucei brucei and T. congolense were compared in vitro. In all the species examined, trypsin levels did not differ significantly up to 24 h post-bloodmeal. There were similar rates of transformation of the bloodstream-form trypanosomes into procyclic (midgut) forms in vivo, so that all species had similar levels of infection in the midgut. However, trypsin levels continued to increase beyond 24 h, reaching a peak between 48 and 72 h. The peak was lowest in G. m. morsitans. The midgut homogenates in this species also had the lowest levels of lectin. The species had the highest levels of mature T. congolense and T. brucei infections. We propose that the lower levels of peak midgut tryspin and lectin in G. m. morsitans is important in the establishment of trypanosome infections in this species of tsetse.RésuméNous avons déterminé les teneurs en trypsine et en lectine de l’intestin moyen de trois espèces de mouche tsétsé, à savoir Glossina morsitans morsitans, G. longipennis et G. fuscipes fuscipes. Nous avons en outre testé in vitro, les capacités de transformation des formes sanguines de Trypanosoma brucei brucei et T. congolense par les homogénats de l’intestin moyen. Chez toutes les espèces étudiées, les teneurs en trypsine ne diffèrent pas significativement pendant les 24 heures qui suivent la prise de sang. In vivo, on observe des taux similaires de transformations des formes sanguines des trypanosomes en formes procycliques (intestin moyen), suggérant que les trois espèces ont des niveaux d’infection comparables dans l’intestin moyen. Cependant, les teneurs en trypsine continuent à augmenter au-delà de 24 h et atteignent un pic entre 48 et 72 h. Le pic est plus bas chez G. m. morsitans. Chez cette espèce, nous constatons que les homogénats d’intestin moyen présentent les concentrations les plus faibles en lectine, alors que nous observons les plus fortes infections de formes mûres de T. congolense et T. brucei. Ceci suggère qu’une faible valeur des pics de concentration en trypsine et en lectine de l’intestin moyen de G. m. morsitans est un facteur important pour l’établissement des infections de trypanosomes.

Biodegradability of polyethylene by bacteria and fungi from Dandora dumpsite Nairobi-Kenya

This study aimed at isolating and identifying bacteria and fungi with the capacity to degrade low density polyethylene (LDPE). The level of biodegradation of LDPE sheets with bacterial and fungal inoculums from different sampling points of Dandora dumpsite was evaluated under laboratory conditions. Incubation of the LDPE sheets was done for sixteen weeks at 37°C and 28°C for bacteria and fungi respectively in a shaker incubator. Isolation of effective candidates for biodegradation was done based on the recorded biodegradation outcomes. The extent of biodegradation on the polyethylene sheets was assessed by various techniques including weight loss analysis, Fourier Transform Infrared Spectroscopy (FTIR) and GC-MS. Fourier Transform Infra-Red spectroscopy (FTIR) analysis revealed the appearance of new functional groups attributed to hydrocarbon degradation after incubation with the bacteria and fungi. Analysis of the 16S rDNA and 18S rDNA sequences for bacteria and fungi respectively showed that bacteria belonging to genera Pseudomonas, Bacillus, Brevibacillus, Cellulosimicrobium, Lysinibacillus and fungi of genus Aspergillus were implicated as polyethylene degraders. An overall analysis confirmed that fungi are generally better degraders of polyethylene than bacteria. The highest fungal degradation activity was a mean weight reduction of 36.4±5.53% attributed to Aspergillus oryzae strain A5, 1 (MG779508). The highest degradation activity for bacteria was a mean of 35.72± 4.01% and 20.28± 2.30% attributed to Bacillus cereus strain A5,a (MG645264) and Brevibacillus borstelensis strain B2,2 (MG645267) respectively. Genus Aspergillus, Bacillus and Brevibacillus were confirmed to be good candidates for Low Density Poly Ethene bio-degradation. This was further confirmed by the appearance of the aldehyde, ether and carboxyl functional groups after FTIR analysis of the polythene sheets and the appearance of a ketone which is also an intermediary product in the culture media. To improve this degrading capacity through assessment of optimum conditions for microbial activity and enzyme production will enable these findings to be applied commercially and on a larger scale.

Genetically Determined Response to Artemisinin Treatment in Western Kenyan Plasmodium falciparum Parasites.

Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya.

Biochemical changes in developing embryos of Schistocerca gregaria (Orthoptera: Acrididae) induced by pheromone produced by ovipositing gregarious females

Trans-generational transfer of gregarious-phase traits in the desert locust Schistocerca gregaria (Forskål, 1775) is mediated by primer gregarizing pheromonal signals produced by ovipositing females that experience crowding. We monitored time-course proteomic events in eggs from solitary-reared locusts that had been exposed for 1, 3, 5, 7, 10 and 12 days to different levels of the sand-associated gregarizing signal originating from 0, 3, 5 or 10 ovipositions by crowd-reared females. Evidence for the phase transition was sought by comparing the protein patterns of embryos thus exposed with those from crowd-reared (gregarious) controls; this comparison was continued until the stage of the first instars. Expressed proteins were analysed by two-dimensional protein gel electrophoresis, and patterns from the different treatments within stages were compared by profile matching and χ2 analyses. Eggs derived from crowd- and solitary-reared females showed essentially similar protein patterns at early stages of embryogenesis; however, mature stages (particularly, days 10 and 12) and hatchlings demonstrated significantly different patterns. Protein patterns of eggs from solitary-reared females that were incubated in sand contaminated with the pheromonal signal and of the hatchlings that emerged were similar to those derived from gregarious females and dependent on the level of the pheromone to which the embryos had been exposed. The results confirm the gregarizing effect of the signal and constitute a useful basis for unravelling the mechanism of the signalling cascades associated with gene expressions triggered by the pheromone.