Relationships between host blood factors and proteases in Glossina morsitans subspecies infected with Trypanosoma congolense.

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.

The influence of host blood on infection rates in Glossina morsitans sspp. infected with Trypanosoma congolense, T. brucei and T. simiae.

Trypanosoma congolense, T. brucei and T. simiae isolated from wild-caught Glossina pallidipes were fed to laboratory-reared G. morsitans centralis and G.m. morsitans to determine the effect of host blood at the time of the infective feed on infection rates. Bloodstream forms of trypanosomes were membrane-fed to flies either neat, or mixed with blood from cows, goats, pigs, buffalo, eland, waterbuck and oryx. The use of different bloods for the infective feed resulted in differences in infection rates that were repeatable for both tsetse subspecies and most parasite stocks. Goat, and to a lesser extent, pig blood facilitated infection, producing high infection rates at low parasitaemias. Blood from cows and the wildlife species produced low infection rates, with eland blood producing the lowest. Addition of D(+)-glucosamine (an inhibitor of tsetse midgut lectin) increased infection rates in most cases. These results indicate the presence of species-specific factors in blood that affect trypanosome survival in tsetse. In certain hosts, factors actually appear to promote infection. The nature of these factors and how they might interact with midgut lectins and proteases are discussed.

Influence of D(+)-glucosamine on infection rates and parasite loads in tsetse flies (Glossina spp.) infected with Trypanosoma brucei.

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.

Studies on tsetse midgut factors that induce differentiation of blood-stream Trypanosoma brucei brucei in vitro.

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.

Population genetics of Trypanosoma brucei and the epidemiology of human sleeping sickness in the Lambwe Valley, Kenya.

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.

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria.

Mortality in adult tsetse, Glossina morsitans morsitans, caused by Pseudomonas aeruginosa, Serratia marcescens, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis H-14, B. thuringiensis 1, B. thuringiensis 5, B. thuringiensis var. insraelensis, and Providentia rettgeri was determined. When bacteria were smeared on rabbit skin and tsetse allowed to feed only once on the contaminated area, mortality 8 days postingestion was significantly higher (P less than 0.01) in tsetse fed on P. aeruginosa, S. marcescens, B. thuringiensis 1, and P. rettgeri and increased when tsetse were allowed to feed for the second time on the contaminated skin. With this smear technique, however, mortalities were generally not remarkable. In artificial membrane feeding experiments using low concentrations of bacteria (-10(6)/ml of blood), the B. thuringiensis strains caused low mortality, except B. thuringiensis H-14, which caused 59% mortality. However, at this concentration, P. aeruginosa, S. marcescens, B. cereus, and P. rettgeri caused highly significant (P less than 0.01) mortality (64-96%). When higher concentrations of bacteria (10(7)/ml) were used, all the bacteria tested, except B. sphaericus, caused high mortality ranging from 70 to 98%. Thus, mortality depended on the species of bacteria, the dose ingested, and time postingestion.

The humoral defense system in tsetse: differences in response due to age, sex and antigen types.

Inoculation of live Escherichia coli into tsetse flies, Glossina morsitans morsitans, stimulated a higher antibacterial immune response in females than in males. It increased with age in females from emergence to approximately 2 weeks and thereafter declined. In males, there was also a significant decrease in immune response with aging. Inoculation of killed bacteria failed to stimulate antibacterial activity but stimulated a lysozyme response which was weaker than that stimulated by live bacteria. No antibacterial activity was present in the hemolymph of larvae from immunized pregnant tsetse. Inoculation of live Trypanosoma brucei brucei and T. congolense failed to induce production of antibacterial activity and lysozyme. Furthermore, tsetse inoculated with or naturally infected with T. b. brucei and T. congolense failed to show any evidence of immunosuppression when challenged with live E. coli. Various species of live bacteria stimulated different levels of antibacterial factors, with Enterobacter cloacae stimulating the highest level of antibacterial activity and E. coli the highest level of lysozyme. Saline in which certain species of bacteria and T. b. brucei were incubated inactivated tsetse immune hemolymph.

The investigation of Trypanosoma brucei isolates obtained from Glossina pallidipes in South Nyanza, Kenya.

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.

The effect of Trypanosoma brucei infection of the localization of salivary gland cholinesterase in Glossina morsitans morsitans.

When salivary glands of the tsetse fly, Glossina morsitans morsitans, are stained for cholinesterase (ChE) activity, a net-like pattern of reaction product is observed surrounding each epithelial cell of the gland's secretory region. Glands infected with Trypanosoma brucei brucei show a progressive reduction in this ChE activity as the parasites develop. When the infection is mature, ChE is rarely detected in the epithelial layer but appears in the lumen of gland. The luminal ChE responds to substrates and inhibitors in the same manner as the epithelium-associated enzyme and appears to have leaked from the epithelium due to cellular damage in epithelium of the infected gland. The possible effect of glandular damage on feeding behaviour and state of health is discussed.

Defence reactions of Glossina morsitans morsitans against different species of bacteria and Trypanosoma brucei brucei.

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.

Characterization of potentially man-infective Trypanosoma brucei from an endemic area of sleeping sickness in Kenya.

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.

Some observations on factors associated with the development of Trypanosoma brucei brucei infections in Glossina morsitans morsitans.

The susceptibility of Glossina morsitans morsitans to Trypanosoma brucei brucei infection was shown to be age-dependent during the first 12 h: the youngest age group (1-8 h after emergence) being more susceptible than the older ones. The susceptibility was enhanced by cooling the young flies to a temperature of 0-5 degrees C for 30 min. Male flies were found to be more susceptible than females. The number of trypanosomes ingested did not influence the subsequent salivary-gland infection rates observed in G.m. morsitans; however, there was a relationship between the number ingested and subsequent T.b. brucei midgut infections in the flies.

The abundance of pathogenic African trypanosomes in the salivary secretions of wild Glossina pallidipes.

Trypanosome infection rates in wild Glossina pallidipes from the Lambwe Valley, Kenya, were determined using salivation and fly dissection methods. Of the 416 flies examined by salivation, one was infected with Trypanosoma brucei, seven with T. congolense, 18 with T. vivax and three with both T. congolense and T. vivax. The fly infected with T. brucei always secreted saliva heavily infected with trypanosomes. Flies infected with T. vivax secreted saliva which contained very few trypanosomes and some of these flies produced negative saliva for several successive days. There was no significant difference between the proportions of male and female flies which were infected with any one species of trypanosome, but there were highly significant differences between the mean numbers of the different species of trypanosome in the saliva.

Trypanosoma brucei: in vitro propagation of metacyclic forms derived from the salivary glands of Glossina morsitans.

1 Metacyclic forms of Trypanosoma brucei obtained from the salivary glands of the tsetse fly, Glossina morsitans have been cultured for the first time in their infective forms for more than 200 days in continuous culture. The parasites were grown at 25 C and 30 C on a bovine embryonic spleen (BESP) feeder layer in buffered RPMI 1640 medium supplemented with 20% heat-inactivated bovine fetal serum (BFS) and 5% lactalbumin hydrolysate. Initial growth rate was enhanced when normal, noninfected, salivary glands were added to the cultures. The parasites thus cultured appeared like slender or intermediate blood stream forms which were infective to rats and mice. Addition of rat anti-T. brucei specific antiserum to the cultures caused agglutination of the parasites and rendered them noninfective. This study opens up new areas of investigating sleeping sickness. The cultured metacyclic parasites have the potential of being applied as antigens for controlling African trypanosomiasis.

Sleeping sickness: in vitro cultivation of Trypanosoma brucei from the salivary glands of Glossina morsitans.

Two strains of Trypanosoma brucei were propagated from the salivary glands of 5 Glossina morsitans for more than 200 days on a bovine embryonic spleen feeder layer using buffered RPMI 1640 medium supplemented with 20% bovine fetal serum. In the first 2 to 3 weeks of cultivation the density of parasites in the salivary glands and culture medium remained constant probably because of defective binary fission. The parasites were infective to rodents only on days 17 and 25. Electron microscopic examination of the parasites on 6 different occasions revealed that they were similar to the immature metatrypomastigotes of T. brucei described in the salivary glands of infected tsetse flies.

Trypanosoma brucei-cultivation in vitro of infective forms derived from the midgut of Glossina morsitans.

Infective forms of Trypanosoma brucei derived from the midgut of Glossina morsitans, have been propagated in vitro for 61 days on a bovine embryonic spleen (BESP) feeder layer using RPMI 1640 medium. It was reproducibly shown that only parasites cultured from the midgut of tsetse flies 12-14 hr after feeding on infected animals could be established in vitro. Cultures thus established were infective to rats and tsetse flies. Only midgut vector types of the parasites were identified by light and electron microscopic techniques.

Development of Trypanosoma (Trypanozoon) brucei in Glossina morsitans inoculated into the tsetse haemocoel.

Classically, infective development of Trypanosoma (Trypanozoon) brucei in tsetse flies is thought to take the route crop-midgut-hindgut proventriculus-hypopharynx-salivary gland, where the parasites reach their infective phase. It has been shown experimentally that T. (T.) brucei is capable of developing up to the infective stage in G. morsitans following inoculation of bloodstream form trypanosomes into the haemocoel. The rabbit on which flies were maintained became infected 18 days after exposure to the bite of experimentally inoculated flies. The possibility that T. (T.) brucei may be transmitted cyclically from tsetse flies to a mammalian host without necessarily following the classical, prescribed route is discussed. Apart from the normal longitudinal binary fission, various modes of multiplication were observed among trypanosomes in the haemocoel, modes which have not been observed previously in the tsetse fly.