PROF. OCHANDA JAMES O.

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.

Bacillus thuringiensis is the most used biological control agent to date. Among major constraints to maize production, safety and hence food sufficiency in Kenya is infestation, damage and contamination by insect pests. Maize grains are adversely damaged by Prostephanus truncatus which occasionally paves way for the growth of aflatoxin producing fungi. The focus of this study was to establish the toxicity of native Bt against adult P. truncatus, second instar larvae of Chilo partellus, Aspergillus flavus and Aspergillus parasiticus. Seven Bt isolates (i.e. KG 411, KG 12-0, KG 20, KG 420, KM 31, KM 12 and KM 24) caused over 50% mean mortality of P. truncatus at the first preliminary dose of 10mg/ml. With subsequent analysis of the efficacy of Bt against P. truncatus, isolate KG 411 was significantly more toxic to it at 95% confidence limit (p <0.001) than all the other Bt isolates. Bt isolate KG 411 had LD50 of 0.30mg/ml which caused 77.1% mean mortality of adult P. truncatus. Potency tests of the Bt isolates against the second instar larvae of C. partellus showed significant differences at 95% confidence limit (p<0.001), with isolate KM 12 causing the highest mean mortality of 76%. Evaluation of effects of spores and crystals produced by the Bt isolates on A. flavus and A. parasiticus showed that isolate KM 31 caused the highest inhibition of fungal growth. Only isolate KM 31 was potent against both P. truncatus and the two fungal species. However isolate KG 411 which was highly toxic against P. truncatus had no significant growth inhibition effect against the two fungal strains. This result demonstrates that native Bt could constitute an alternative biological control option for management of adult P. truncatus, A. parasiticus and A. flavus in maize protection.

Bacillus thuringiensis (Bt) delta endotoxins represent the most successful use of biological control agents targeting crop pests to date. Studies have shown that Bt produces a wider range of toxins targeting a variety of unrelated pests than was initially documented. This further increases the prospects for its wider use as a microbial biopesticide. In this view the study sought to isolate and characterize native Bt from two different ecological regions; Kenya coastal intertidal brackish sediments and farmlands in Machakos district. Twenty eight Bt isolates were isolated and identified. Distinctions between the isolates were based on their morphological appearances, presence of parasporal inclusions and biochemical characteristics. Results for staining tests revealed that the isolates were Gram positive, rod shaped cells, with the spores terminally located. The cells from the two ecological regions did not have significant variation in average size. The rods had length ranging between 2.0 μm and 3.0 μm on average while the width was approximately 1 μm on average. Parasporal inclusions had varied shapes (i.e. bipyramidal, circular, squared, oval and rhomboid). Both biochemical tests (Proskauer (VP) test and catalase test) were positive. Average optimum temperature range for growth was between 28°C and 35°C while the optimal pH growth range was between 5.5 and 7.5. Both the morphological and biochemical results provided evidence that the isolates were different forms of Bacillus thuringiensis. Out of the fifty six soil and sediment samples, 28 samples (50%) yielded Bacillus thuringiensis. 53.6% of the isolates were recovered from samples from farms in Machakos district and 46.4% from the intertidal brackish sediments from the Gazi coastal mangroves of Kenya. This shows that Bt is also present in soils which are under constant cultivation and those lying fallow within the intertidal brackish sediments

BACKGROUND: Malaria control in Africa relies primarily on early effective treatment for clinical disease, but most early treatments for fever occur through self-medication with shop-bought drugs. Lack of information to community members on over-the-counter drug use has led to widespread ineffective treatment of fevers, increased risks of drug toxicity and accelerating drug resistance. We examined the feasibility and measured the likely impact of training shop keepers in rural Africa on community drug use. METHODS: In a rural area of coastal Kenya, we implemented a shop keeper training programme in 23 shops serving a population of approximately 3500, based on formative research within the community. We evaluated the training by measuring changes in the proportions of drug sales where an adequate amount of chloroquine was purchased and in the percentage of home-treated childhood fevers given an adequate amount of chloroquine. The programme was assessed qualitatively in the community following the shop keeper training. RESULTS: The percentage of drug sales for children with fever which included an antimalarial drug rose from 34.3% (95% CI 28.9%-40.1%) before the training to a minimum of 79.3% (95% CI 71.8%-85.3%) after the training. The percentage of antimalarial drug sales where an adequate amount of drug was purchased rose from 31.8% (95% CI 26.6%-37.6%) to a minimum of 82.9% (95% CI 76.3%-87.3%). The percentage of childhood fevers where an adequate dose of chloroquine was given to the child rose from 3.7% (95% CI 1.2%-9.7%) before the training to a minimum of 65.2% (95% CI 57.7%-72.0%) afterwards, which represents an increase in the appropriate use of over-the-counter chloroquine by at least 62% (95% CI 53.7%-69.3%). Shop keepers and community members were strongly supportive of the aims and outcome of the programme. CONCLUSIONS: The large shifts in behaviour observed indicate that the approach of training shop keepers as a channel for information to the community is both feasible and likely to have a significant impact. Whilst some of the impact seen may be attributable to research effects in a relatively small scale pilot study, the magnitude of the changes support further investigation into this approach as a potentially important new strategy in malaria control.

BACKGROUND: Malaria control in Africa relies primarily on early effective treatment for clinical disease, but most early treatments for fever occur through self-medication with shop-bought drugs. Lack of information to community members on over-the-counter drug use has led to widespread ineffective treatment of fevers, increased risks of drug toxicity and accelerating drug resistance. We examined the feasibility and measured the likely impact of training shop keepers in rural Africa on community drug use. METHODS: In a rural area of coastal Kenya, we implemented a shop keeper training programme in 23 shops serving a population of approximately 3500, based on formative research within the community. We evaluated the training by measuring changes in the proportions of drug sales where an adequate amount of chloroquine was purchased and in the percentage of home-treated childhood fevers given an adequate amount of chloroquine. The programme was assessed qualitatively in the community following the shop keeper training. RESULTS: The percentage of drug sales for children with fever which included an antimalarial drug rose from 34.3% (95% CI 28.9%-40.1%) before the training to a minimum of 79.3% (95% CI 71.8%-85.3%) after the training. The percentage of antimalarial drug sales where an adequate amount of drug was purchased rose from 31.8% (95% CI 26.6%-37.6%) to a minimum of 82.9% (95% CI 76.3%-87.3%). The percentage of childhood fevers where an adequate dose of chloroquine was given to the child rose from 3.7% (95% CI 1.2%-9.7%) before the training to a minimum of 65.2% (95% CI 57.7%-72.0%) afterwards, which represents an increase in the appropriate use of over-the-counter chloroquine by at least 62% (95% CI 53.7%-69.3%). Shop keepers and community members were strongly supportive of the aims and outcome of the programme. CONCLUSIONS: The large shifts in behaviour observed indicate that the approach of training shop keepers as a channel for information to the community is both feasible and likely to have a significant impact. Whilst some of the impact seen may be attributable to research effects in a relatively small scale pilot study, the magnitude of the changes support further investigation into this approach as a potentially important new strategy in malaria control.

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.

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.

The midgut of the human body louse Pediculus humanus humanus contains a thermally stable leucine aminopeptidase, which was detected by agarose gel electrophoresis using l-amino oxidase. Midgut extracts were homogenized in saline or in 1% Triton X-100 and the aminopeptidase was purified by Superose 6 gel filtration chromatography. A peak with enzyme activity that was extracted with or without Triton X-100 was eluted at a molecular weight 67–69 kDa. Non-denaturing polyacrylamide gel electrophoresis resolved one band of molecular weight of 69 kDa for samples that were extracted in a saline buffer. Two closely linked bands of molecular weight 67 kDa and 69 kDa were observed in samples that were extracted in 1% Triton X-100.

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.

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.

A leucine aminopeptidase was found in the midgut of the human body louse, Pediculus humanus humanus L. (Anoplura: Pediculidae). The enzyme is activated by the bloodmeal with a pH optimum at 8. The enzyme is soluble in both aqueous and detergent-containing solutions. The two forms of the enzyme had the same Km but exhibited different catalytic activities with regard to Vmax values in these solutions. The enzyme is inhibited competitively by a substrate analogue 1,10-phenanthroline and by Mn2+ ions in the presence and absence of detergent.

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.

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.

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.

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.

Immunization of rabbits with a faecal extract of the human body louse (Pediculus humanus) induced a high titre of specific IgG. The mean weight of blood taken by females fed on the immunized rabbits was significantly lower (29%) than taken by females fed on the control rabbits. The mean number of eggs per female fed on the immunized rabbits was significantly lower than for females fed on the control rabbits. The hatchability of the eggs laid by lice fed on immunized rabbits (91%) was significantly lower than of those fed on control rabbits (94%). The rate of development of nymphs fed on control rabbits was significantly higher than those fed on the immunized rabbits. There was no difference in survival rates of lice fed on immunized and control rabbits.

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.

Immunization of rabbits with a faecal extract of the human body louse (Pediculus humanus) induced a high titre of specific IgG. The mean weight of blood taken by females fed on the immunized rabbits was significantly lower (29%) than taken by females fed on the control rabbits. The mean number of eggs per female fed on the immunized rabbits was significantly lower than for females fed on the control rabbits. The hatchability of the eggs laid by lice fed on immunized rabbits (91%) was significantly lower than of those fed on control rabbits (94%). The rate of development of nymphs fed on control rabbits was significantly higher than those fed on the immunized rabbits. There was no difference in survival rates of lice fed on immunized and control rabbits.

Immunogenic midgut antigens of the human body louse, Pediculus humanus humanus L., were localized using rabbit antisera against a louse-midgut extract followed by a 2nd antibody conjugated to either fluorescein or colloidal gold. Strong fluorescence was observed on the outer membrane of the epithelial cell of the midgut. The immunogold technique revealed that most of the antigens were localized on the microvilli of the midgut cells. Small numbers of gold particles were also seen in the gut lumen and within the cell cytoplasm. Only a few gold particles were seen in the lumen of the gut sections incubated with control sera.

Immunization of rabbits with a faecal extract of the human body louse (Pediculus humanus) induced a high titre of specific IgG. The mean weight of blood taken by females fed on the immunized rabbits was significantly lower (29%) than taken by females fed on the control rabbits. The mean number of eggs per female fed on the immunized rabbits was significantly lower than for females fed on the control rabbits. The hatchability of the eggs laid by lice fed on immunized rabbits (91%) was significantly lower than of those fed on control rabbits (94%). The rate of development of nymphs fed on control rabbits was significantly higher than those fed on the immunized rabbits. There was no difference in survival rates of lice fed on immunized and control rabbits.

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.

Immunization of rabbits with a faecal extract of the human body louse (Pediculus humanus) induced a high titre of specific IgG. The mean weight of blood taken by females fed on the immunized rabbits was significantly lower (29%) than taken by females fed on the control rabbits. The mean number of eggs per female fed on the immunized rabbits was significantly lower than for females fed on the control rabbits. The hatchability of the eggs laid by lice fed on immunized rabbits (91%) was significantly lower than of those fed on control rabbits (94%). The rate of development of nymphs fed on control rabbits was significantly higher than those fed on the immunized rabbits. There was no difference in survival rates of lice fed on immunized and control rabbits.

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 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 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 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 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 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.

Abstract in proceedings of Biotech SA 93 held at Rhodes University, 31 January-3 February 1993

Abstract in proceedings of Biotech SA 93 held at Rhodes University, 31 January-3 February 1993

1. Larval development in Glossina species occurs in utero with the mature third instar larva being deposited after a developmental period of 7 days. 2. In this study, the patterns of cuticular protein synthesis during larval development were analysed by two-dimensional gel electrophoresis. 3. From the results, four types of cuticle proteins were identified: those specific to larval, pupal and adult cuticles, and others common to all the stages. 4. Few cuticular proteins were synthesized between the first and second larval instars. By the third larval instar (two days before larviposition), a large number of proteins (Mr < or = 30 kDa) were induced. These proteins persisted up to the brown pupal stage and showed a rapid decline thereafter. Most of the proteins with molecular weights Mr < or = 30 kDa were undetectable at apolysis (5 days after larviposition). 5. By day 15 of the pupal stage, the number of cuticle proteins was very small. The protein profile during the pupal stages remained relatively constant. This was probably due to the fact that the pupal cuticle does not provide any protection since it is itself enclosed at all times within the protective puparium.

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 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 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.

1. Larval development in Glossina species occurs in utero with the mature third instar larva being deposited after a developmental period of 7 days. 2. In this study, the patterns of cuticular protein synthesis during larval development were analysed by two-dimensional gel electrophoresis. 3. From the results, four types of cuticle proteins were identified: those specific to larval, pupal and adult cuticles, and others common to all the stages. 4. Few cuticular proteins were synthesized between the first and second larval instars. By the third larval instar (two days before larviposition), a large number of proteins (Mr < or = 30 kDa) were induced. These proteins persisted up to the brown pupal stage and showed a rapid decline thereafter. Most of the proteins with molecular weights Mr < or = 30 kDa were undetectable at apolysis (5 days after larviposition). 5. By day 15 of the pupal stage, the number of cuticle proteins was very small. The protein profile during the pupal stages remained relatively constant. This was probably due to the fact that the pupal cuticle does not provide any protection since it is itself enclosed at all times within the protective puparium.

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.

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.

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.

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.

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.

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.

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.

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.

The toxin produced by Clostridium botulinum type C 6813 (C-6813) was purified 1,009-fold from the culture supernatant in an overall yield of 30%. The specific toxicity was 1.1 X 10(7) mouse minimum lethal doses per mg of protein. The toxin had a molecular weight of 144,000, composed of the light and heavy chains with molecular weights of 52,000 and 92,000, respectively, linked by one or two disulfide bond(s). The purified C-6813 toxin heavy and light chains reacted strongly with anti-type D heavy chain immunoglobulin G and anti-type C1 light chain immunoglobulin G, respectively. The amino acid compositions of C-6813 toxin heavy and light chains were more similar to those of type D heavy chain and type C1 light chain than to those of type C1 heavy chain and type D light chain, respectively. These results suggest that in the toxin produced by the type C strain at least two subtypes exist.

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.

The toxin produced by Clostridium botulinum type C 6813 (C-6813) was purified 1,009-fold from the culture supernatant in an overall yield of 30%. The specific toxicity was 1.1 X 10(7) mouse minimum lethal doses per mg of protein. The toxin had a molecular weight of 144,000, composed of the light and heavy chains with molecular weights of 52,000 and 92,000, respectively, linked by one or two disulfide bond(s). The purified C-6813 toxin heavy and light chains reacted strongly with anti-type D heavy chain immunoglobulin G and anti-type C1 light chain immunoglobulin G, respectively. The amino acid compositions of C-6813 toxin heavy and light chains were more similar to those of type D heavy chain and type C1 light chain than to those of type C1 heavy chain and type D light chain, respectively. These results suggest that in the toxin produced by the type C strain at least two subtypes exist.

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.