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Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies. The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0. 0001) than those of the controls. Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Chi2 = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates. Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells.

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Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P < 0.0001) and produced pupae that were of significantly less weight in milligrams (P < 0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P < 0.00001).

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Host blood effects on Trypanosoma congolense establishment in Glossina morsitans morsitans and Glossina morsitans centralis were investigated using goat, rabbit, cow and rhinoceros blood. Meals containing goat erythrocytes facilitated infection in G.m.morsitans, whereas meals containing goat plasma facilitated infection in G.m.centralis. Goat blood effects were not observed in the presence of complementary rabbit blood components. N-acetyl-glucosamine (a midgut-lectin inhibitor) increased infection rates in some, but not all, blood manipulations. Cholesterol increased infection rates in G.m.centralis only. Both compounds together added to cow blood produced superinfection in G.m.centralis, but not in G.m.morsitans. Midgut protease levels did not differ 6 days post-infection in flies maintaining infections versus flies clearing infections. Protease levels were weakly correlated with patterns of infection, but only in G.m.morsitans. These results suggest that physiological mechanisms responsible for variation in infection rates are only superficially similar in these closely-related tsetse.

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Relatively simple protocols employing non-radioactive DNA probes have been used for the detection of African trypanosomes in the blood of mammalian hosts and the saliva of live tsetse flies. In combination with the polymerase chain reaction (PCR), the protocols revealed trypanosomes in buffy-coat samples from antigenaemic but aparasitaemic cattle and in the saliva of live, infected tsetse flies. Furthermore, the protocols were used to demonstrate concurrent natural infections of single tsetse flies with different species of African trypanosomes.

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The pattern of infection in Glossina morsitans morsitans and G. m. centralis membrane-fed on eland, buffalo or goat blood mixed with Trypanosoma congolense or T. brucei was studied from day 1 to day 10. Tsetse were initially permissive vectors, with most flies harbouring infections of 10(4)-10(5) parasites on day 3. However, after a second blood meal on day 3, flies cleared many infections, with G. m. morsitans clearing more infections than G.m. centralis. Infective feeds of goat blood consistently increased final infection rates by limiting the number of infections lost between days 3 and 6. In further experiments with G. m. morsitans only, this effect was replicated by feeding flies on erythrocytes but not on serum. These results suggest that compounds from some mammalian erythrocytes match the target specificity of G. m. morsitans midgut lectins and, hence, have a protective effect on trypanosome establishment in the fly.

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Teneral Glossina morsitans centralis, G. m. morsitans and G. pallidipes were infected with three different clones of Trypanosoma brucei in blood containing D(+)-glucosamine, an inhibitor of tsetse midgut lectin. On average, 5 days of D(+)-glucosamine treatment tripled infection rates, without affecting the proportion of infections that matured. Total infection rates were equal in males and females, but twice as many infections matured in males. Counts of parasites in the guts and salivary glands of 277 flies revealed order of magnitude differences among flies, with females consistently having 2-3-times as many parasites as males. Parasite numbers varied in a sex-specific manner among tsetse-clone combinations, but these differences were not correlated with similar large differences in infection rates. D(+)-glucosamine treatment had no significant effect on parasite loads.

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An in vitro system for studying the transformation of bloodstream forms of Trypanosoma brucei brucei into procylic (midgut) forms is described. In this system, transformation of the parasites was stimulated by Glossina morsitans morsitans midgut homogenates at 27 degrees C but not at 4 degrees C. The transformation-stimulating capacity was irreversibly destroyed by heating the midgut homogenates at 60 degrees C for 1 h. A correlation was established between the transformation activity of the midgut homogenates and trypsin activity. The protease inhibitors (soybean trypsin inhibitor and N-p-tosyl-L-lysine-chloromethyl-ketone) inhibited trypsin activity and completely blocked the transformation of the parasites. Furthermore, the midgut homogenates could induce transformation only in the presence of blood. These results provide evidence for the involvement of trypsin or trypsin-like enzymes within the tsetse midgut in stimulation of the transformation of bloodstream trypanosomes.

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1. The major protein in the milk gland secretions of the tsetse fly, Glossina morsitans morsitans, was isolated by a combination of gel permeation chromatography and crystallization. 2. It has a native Mr approximately 47,000 and is composed of two identical polypeptide chains (Mr approximately 21,000) as determined by chemical cross-linking studies. The protein has no covalently-bound carbohydrates or lipids. Amino acid analysis of the protein revealed relatively high amounts of the aromatic amino acids, tyrosine (9.1 mol.%) and phenylalanine (8.5 mol.%). Immunoblotting experiments using antiserum against the protein revealed no cross-reactivity with any other milk proteins. 3. Quantitation of the protein during the pregnancy cycle showed that synthesis of the protein by the milk glands of adult female flies starts as the larva moults into second instar and rapidly declines as it matures into third instar. 4. It is proposed that the major milk gland protein could provide essential amino acids needed for the puparium formation.

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The utility of recombinant DNA probes in the detection of natural trypanosome infection of tsetse flies has been assessed in Lambwe Valley, near the shores of Lake Victoria, Kenya. The tsetse flies were surveyed during two different seasons in 1988. Three different probes used each contained highly repetitive DNA sequences specific for a species or subspecies of trypanosomes of the Nanomonas subgenus. A fourth probe contained repetitive sequences common to trypanosome species of the Trypanozoon subgenus. Mixed mature or immature infections were detected in a variety of combinations in different individual tsetse flies. Such infections were detected in both the guts and mouthparts of some tsetse flies. Simultaneous natural infection of tsetse with the savannah type Trypanosoma congolense and Kilifi type T. congolense, T. congolense and Trypanosoma brucei or T. congolense and Trypanosoma simiae were demonstrated. The probes have thus been used to demonstrate the presence of Lambwe Valley, south-western Kenya, of a type of T. congolense first observed among trypanosome isolates obtained from sentinel cattle exposed to natural infection on a ranch at Kilifi on the Kenya coast. This type of T. congolense appears not to be confined to the coastal region nor to any particular species of tsetse flies and may contribute significantly to livestock morbidity in other areas of eastern Africa. In the Kilifi area, T. congolense was found primarily in Glossina austeni; in Lambwe valley, it was found in Glossina pallidipes.

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Numerical taxonomy was used to review isoenzyme variation in isolates of Trypanosoma brucei obtained from cattle, tsetse, humans and wildlife from the Lambwe Valley, Kenya. From isoenzyme information alone, it was possible to classify isolates as to source through the use of linear discriminant functions analysis, with an error rate of only 2% in humans, and 14% over all groups. Differentiation was mostly dependent on patterns in the enzymes ASAT, PEP1, and ICD. Parasites from non-human sources, especially tsetse, were characterized by high isoenzyme diversity, and many unique zymodemes. Observed frequencies of genotypes for ICD, ALAT, and ASAT did not agree with expected frequencies based on random mating of a diploid organism. Deviations were particularly large for tsetse isolates, and were mostly due to a deficiency of one homozygote. Cluster analysis revealed complex relationships among isolates, with patterns evolving through time. Major human zymodemes from the 1970s clustered together with most wildlife isolates from East Africa. This chronic human-wildlife transmission cycle was characterized by ASAT pattern I. Other, minor human zymodemes were associated with a human-cattle transmission cycle characterized by ASAT pattern VII. These original chronic transmission cycles appeared to change in 1980 with the appearance of two new zymodemes in humans. These zymodemes involved changes in ALAT and/or PGM to patterns typical of tsetse and cattle isolates. A resultant epidemic was halted with repeated aerial spraying of endosulfan in 1981. Since then, a variety of new zymodemes of unknown human infectivity have appeared. The origins of these changes are discussed in terms of genetic exchange in tsetse, adaptation to human and cattle transmission cycles, and selection resulting from chronic use of insecticides.

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A drug-resistant Trypanosoma congolense strain with predetermined curative doses (CD50 and CD90) of samorin at 13.9 +/- 1.02 and 20.3 +/- 1.13 mg/kg body weight, respectively, was cyclically transmitted through tsetse flies and by syringe passages in mice in the absence of drug pressure. The changing levels of drug sensitivity were determined after every 3rd cyclic and 5th syringe passage intervals. It was noted that when the strain was maintained in tsetse flies through 12 cyclical transmissions, the CD50 and CD90 dropped slightly from 13.9 to 11.9 +/- 1.06 and from 20.3 to 18.0 +/- 1.08 mg/kg body weight, respectively. This decrease in the level of resistance was not significant (P greater than 0.05). However, when the trypanosomes were maintained by syringe passages in mice, there was a significant reduction (P less than 0.05) in the degree of resistance (CD50 from 13.9 to 11.4 +/- 1.07 and CD90 from 20.3 to 16.7 +/- 1.16 mg/kg), by the 15th syringe passage.

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A drug-sensitive Trypanosoma congolense (IL 1180 strain), with a known CD50 and CD90 (doses required to cure 50 and 90% of the infected animals) was cyclically passaged through tsetse flies. The infected flies were then fed on rabbits which received weekly prophylactic treatment of Samorin. It was observed that the infections arising from flies maintained for over 60 days on drug-treated rabbits required higher curative doses to achieve a 50 and 90% cure. The results of this work suggest that a selection for drug resistance occurs when trypanosome stage in Glossina is continuously exposed to drug-treated animals.

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During studies to determine the main Trypanosoma brucei rhodesiense transmission sites in Lambwe Valley, Western Kenya, Glossina pallidipes were collected from two areas in the valley and examined for trypanosome infection. T. brucei isolated from infected flies were tested for their response to the lethal effects of human blood (Blood Incubation Infectivity Test, BIIT) and also characterized using isoenzyme electrophoresis. Six of the 26 T. brucei tested were BIIT positive, two of which had enzyme profiles identical to human isolates. The 26 isolates were grouped into 10 zymodemes. Two zymodemes were identical to T. b. rhodesiense isolated from sleeping sickness patients, one of which was identical to the predominant zymodemes among patients from Lambwe Valley area. The other zymodeme was identical to an isolate from a patient from the Busoga (Uganda) sleeping sickness epidemic focus. These two Zymodemes were BIIT positive. It is suggested that there has been an exchange of human infecting organisms between the Kenya and the Uganda foci.

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Tsetse flies, Glossina morsitans morsitans, fed on rats infected with Trypanosoma brucei brucei showed wide fluctuations in total and differential haemocyte counts. Similar fluctuations occurred in controls fed on non-infected rats and also between the two groups without showing any difference which could be attributed to the infection. Trypanosome infection of the tsetse haemocoel occurred in 16.25% of the flies, starting from the second day after feeding on the infected rats, but salivary glands and proboscis became infected only after the eleventh day. About 2% of bloodstream forms of T. b. brucei injected into tsetse haemocoels completed their developmental cycle successfully. Injection of tsetse homogenates into teneral G. m. morsitans prior to exposure to trypanosome-infected feed increased T. b. brucei infections in the flies significantly. Injection of live Escherichia coli, Enterobacter cloacae and Acinetobacter calcoaceticus into tsetse induced a remarkable increase in two pre-existing haemolymph proteins with molecular weights of about 70 and 17 kilodaltons, while live Bacillus subtilis and Micrococcus luteus induced a very weak response or sometimes none at all. T. b. brucei also failed to induce any increase in these proteins. Inoculation of G. m. morsitans with live E. coli und T. b. brucei prior to feeding on trypanosome-infected rats had no effect on the salivary gland and proboscis infection rates by T. b. brucei. Injection of live T. b. brucei into the haemocoels of tsetse caused no change in total haemocyte counts, but the trypanosomes disappeared from the haemolymph so rapidly that by 48 h post-injection, only about 1% were left.

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Human trypanosomiasis caused by Trypanosoma brucei rhodesiense has affected the human population in Lambwe Valley, western Kenya, for more than 20 years. A characteristic feature of the disease has been the repeated recrudescense at restricted residual foci. Studies carried out on the incidence of trypanosome infection rates in the vector Glossina pallidipes during the last two years have shown high incidence of pathogenic African trypanosomes in the area. An overall trypanosome infection rate of 18.6% was recorded. T. vivax accounted for 15% of all infected flies whereas T. congolense was detected in 2.7% of flies examined. T. brucei infections were observed in 0.9% of the flies. Twenty-six T. brucei isolates were tested for their sensitivity to human plasma using Blood Incubation Infectivity Test (BIIT), and 14 (54%) gave positive BIIT reactions. Isoenzyme characterisation of all BIIT positive isolates was carried out in order to detect any variations within these potentially man-infective T. brucei. Of the seven zymodemes observed, two were more frequently represented. Zymodeme (Z1) was represented by five stocks. This zymodeme was completely identical electrophoretically to T.b. rhodesiense isolated from patients in the same locality. Zymodeme (Z4) was represented by four stocks. This zymodeme does not have the ALAT I in combination with PGM II and ICD II patterns which are characteristic of typical West African T.b. gambiense. The high frequency of PGM III and ICD III strongly suggested the occurrence of hybridization of zymodemes in the area.

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Topical application of a natural pyrethrum extract on male and pregnant female Glossina morsitans morsitans resulted in higher mortality for flies infected with Trypanosoma brucei brucei than for uninfected control flies. Infected males showed a significantly higher mortality while infected pregnant females showed a marginally significant increase in mortality. Results support the hypothesis that infected flies are less healthy than uninfected flies. Results also parallel previous findings using endosulfan as the topical applicant and exclude the likelihood that the results were because of a peculiar effect of endosulfan.

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