PROF. NGUU EDWARD KINYUA

OBJECTIVE: Screening and biochemical characterisation of trypanosome-lysing factor (trypanolysin) from non-vector insect, Schistocerca gregaria. DESIGN: Laboratory based experiment. SETTING: Department of Biochemistry, University of Nairobi. RESULTS: Lysis of isolated trypanosomes was demonstrated with midgut homogenates of natural vector Glossina morsitans centralis as well in non-vector insects. The highest trypanolytic activity was observed in midgut homogenate of the desert locust. Schistocerca gregaria followed by the cockroach, Periplaneta americana (L). Further studies on the S. gregaria trypanolytic factor showed its proteinaceous nature due to its sensitivity to temperatures above 40 degrees C and to proteases. Additionally, the factor showed lectin-like properties since the activity was blocked by D-glucosamine. CONCLUSION: The trypanolytic factor has the potential of being used to modulate tsetse fly vectorial capacity.

Nonsusceptibility to Bombyx mori densovirus type 2 (BmDNV-2) is controlled by a recessive non-susceptibility gene, nsd-2 (non-susceptibility to DNV-2) in B. mori. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 (BF1) progeny were used for linkage analysis and mapping of nsd-2 using silkworm strains C124 and 902, which are classified as being highly susceptible and non-susceptible to DNV-2, respectively. BF1 larvae were inoculated twice with DNV-2 virus at the first and second instar stages. DNA was extracted from each of the surviving fifth instar larvae and analysed by RFLP inheritance patterns using probes specific to each of the 28 linkage groups of B. mori. Our results indicated that the non-susceptibility gene was linked to linkage group 17, since all surviving larvae showed the homozygous profile of strain 902 in their genotype. The other linkage groups showed mixtures of heterozygous and homozygous genotypes, indicating an independent assortment. A linkage map of 30.6 cM was constructed for linkage group 17 with nsd-2 mapped at 24.5 cM and three closely linked cDNA markers were identified.

Separation of midgut membrane proteins from the tick, Ambylomma variegatum, using a nonionic detergent (Triton X-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (Mr approximately 27,000, 67,000, 86,000 and 95,000,) and 2 proteins (M, approximately 54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins Mr approximately 27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, the reproductive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively.

The spliced leader (SL) RNA plays a key role in mRNA maturation in trypanosomatid protozoa by providing the SL sequence, which is joined to the 5' end of every mRNA. As a first step towards a better understanding of the biogenesis and function of the SL RNA, we expressed a tagged SL RNA gene in a cell-free system of procyclic Trypanosoma brucei cells. Transcription initiates at + 1 can be detected as early as 1 min after addition of extract. Transcription of the SL RNA gene in vitro, as well as in permeable cells, is mediated by an alpha-amanitin/tagetitoxin resistant complex, suggesting a promoter that is intermediate between a classical RNA polymerase II and RNA polymerase III promoter. An analysis of the promoter architecture of the SL RNA gene revealed that regulatory elements are located upstream of the coding region and that the SL sequence, in contrast to the nematode SL sequence, is not required for T. brucei SL RNA gene transcription.

Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

The efficacy of bloodmeal digestion in teneral Glossina morsitans centralis fed on rabbits immunized with tsetse fly midgut extracts was progressively monitored over a period of 96 hours. Flies fed on immunized rabbits showed reduced rate of bloodmeal digestion as compared to the controls. Although there was insignificant difference in the rate of bloodmeal digestion upto 24 hours post-feeding in later stages of digestion there was quite a significant difference. Polyacrylamide gel electrophoretic patterns of bloodmeal drawn from the posterior sections of the midgut demonstrated that, bloodmeal is completely degraded in the midgut after 96 hours in the control flies, while substantial amount is still undigested in the experimental flies. However, not much difference in the rates of digestion was observed with bloodmeal drawn from the anterior section of the midgut. These results suggests that when flies are fed on rabbits immunized with tsetse fly midgut extract, there is an impairment on the efficiency of digestion. The anti-midgut antibodies could be interfering with either the induction or proteolytic activity of the midgut enzymes.

The human body louse, Pediculus humanus, showed eighteen midgut proteins ranging between 12 and 117 kDa, when analysed by SDS-PAGE electrophoresis. Seven of them (12 kDa, 17 kDa, 29 kDa, 35 kDa, 40 kDa, 55 kDa and 97 kDa) were major bands based on their intensity of staining. The immunization of rabbits with a midgut extract elicited the production of protective polyclonal antibodies. These antibodies reacted strongly with all major midgut proteins as well as with 63 kDa and 117 kDa proteins when tested by the Western blot technique. The analysis of the proteins revealed that the 12 kDa, 25 kDa, 29 kDa, 35 kDa, 45 kDa, 87 kDa and 97 kDa proteins are glycosylated and none of them contained a lipid moiety. By electroelution, the proteins of 35 kDa and 63 kDa were purified. On trypsinization, the proteins of 35 kDa and 63 kDa produced four major fragments (F1, F2, F3, and F4) when resolved on a 18% SDS-PAGE. The F1 fragment of the 35 kDa protein reacted with the polyclonal antibodies by the immunoblot technique.

The haemolymph of the tsetse fly, Glossina morsitans morsitans, contains a high (lipophorin) and a low molecular weight protein of high densities, 1.11 and 1.29 g/ml, respectively. The purification of the proteins was achieved by a combination of density gradient ultracentrifugation and reported gel permeation chromatography. The lipophorin is of high molecular weight (M(r) integral of 600,000) and consists of two apoproteins, apolipophorin I (M(r) integral of 250,000) and apolipophorin II (M(r) integral of 80,000) both of which are glycosylated. Lipophorin also has a pI of 6.1. However, electrophoresis under non-denaturing and denaturing conditions showed the low molecular weight protein to be a single polypeptide chain (M(r) integral of 23,000). Amino acid analysis revealed a relatively high content of the acidic amino acids as well as serine and glycine. The protein contained lipids as shown by Sudan Black staining but was unglycosylated. Using rabbit antiserum against the isolated protein in immunodiffusion and immunoblotting experiments, no cross-reactivity was detected with haemolymph samples from insects representing six orders. In conclusion, the finding of lipophorin suggests that, although flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements. However, the results for the low molecular weight protein indicate that the protein is unique to Glossina, suggesting that it may have an important role in the physiology of this insect and is therefore a significant target for vector management.

Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.