Culture form and tsetse fly midgut form procyclic Trypanosoma brucei express common proteins.

Proteins expressed by culture form and tsetse fly midgut form procyclic trypanosomes were examined by polyacrylamide gel electrophoretic techniques. Analysis of the proteins of the two forms of procyclic organisms was performed by comparison of autoradiographs of high resolution two-dimensional polyacrylamide gels prepared using [35S]methionine-labelled parasites. Only eight spots were found to differ between autoradiographs of culture form and tsetse fly midgut form parasites. Seven of these differences were attributable to 35S-labelled non-trypanosomal proteins from the tsetse midgut. The other single spot difference was seen in one of two experiments and was present only in the autoradiograph of the material from trypanosome-infected tsetse fly midgut. Thus the cultivated procyclic organisms did not differ significantly from their tsetse-derived counterparts in protein composition and therefore their use as models for the natural stage is probably justified for most studies.

The chromosome profiles of Trypanosoma congolense isolates from Kilifi, Kenya and their relationship to serodeme identity.

Chromosomal DNA from 117 Trypanosoma congolense clones from 54 stocks, isolated from cattle introduced onto a ranch in Kilifi in the coastal area of Kenya, was fractionated by the orthogonal field alternation gel electrophoresis technique. The technique resolved chromosomes in the size range of 100 kb-1 Mb. The chromosome profile for cloned trypanosome populations was relatively stable with regard to number and size of the chromosome bands following transmission in mice, cattle, goats or tsetse flies. Only in one clone was a shift observed in the position of one medium-sized chromosome band following cyclical development in tsetse. On the basis of their chromosome profiles, the 117 clones could be divided into 18 distinct groups. Representative clones, randomly selected from 7 of the 18 chromosome profile groups were inoculated into steers and goats in order to raise variable antigen type (VAT) repertoire-specific infection sera. Cross-neutralization assays demonstrated that recovery sera from animals infected with a clone neutralized all the clones with an identical chromosome profile. This suggests that clones having an identical chromosome profile also express an identical VAT-repertoire (serodeme).

Absence of a surface coat from metacyclic Trypanosoma vivax: possible implications for vaccination against vivax trypanosomiasis.

Trypomastigotes attached to the wall of the hypopharynx in tsetse flies infected with Trypanosoma vivax are believed to represent the true metacyclic stage of this trypanosome. Electron microscopy demonstrates that attachment is mediated by hemidesmosome-like junctions along the flagellar membrane and that none of the trypomastigotes, either attached or free in the hypopharynx lumen, possesses a surface coat comparable with that on the metacyclics of T. brucei and T. congolense and on the bloodstream stages of all salivarian trypanosomes. As the variable antigen of bloodstream and metacyclic T. brucei is located in the surface coat, the absence of the coat from metacyclic T. vivax suggests that the mechanism of antigenic variation in this species may be somewhat different from that of antigenic variation in T. brucei, and that vaccination of cattle against T. vivax may prove a simpler proposition than vaccination against T. brucei.

Trypanosoma vivax, T. congolense or T. brucei infection rates in Glossina morsitans when maintained in vitro on the blood of goat or calf.

Tenerals of Glossina morsitans morsitans and G. m. centralis were infected with Trypanosoma vivax, T. congolense or T. brucei by feeding mainly on infected goats and then maintained either in vivo on uninfected calves, goats or rabbits, or fed in vitro upon heparinised or defibrinated blood of goats or calves for 21 days for T. vivax and T. congolense and 30 days for T. brucei and then dissected. The observed differences in the infection rates for all three trypanosome species maintained on different diets were small and/or inconsistent and possibly are of no significance. It is therefore likely that the in vitro feeding of the tsetse on these diets after infected blood meal has no adverse effect on the cyclical development of these trypanosome species in these vectors.

Excretion of uric acid and amino acids during diuresis in the adult female Glossina morsitans.

Radiometric analysis was carried out on the urine collected for one hour following feeding of the adult female Glossina morsitans on day 1 of a pregnancy cycle, which had previously received haemocoelic injections of U-14C labelled arginine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine or valine. Mean radioactivity in the urine was quite high after labelled arginine (17.4% of injected activity) and histidine (21.8%) administration, most of the activity being in the amino acid fractions. With the remaining six labelled amino acids, mean radioactivity in the urine varied between 1.6 and 7.2% of injected activity, most of this activity occurred in a non-amino acid fraction (probably uric acid), though low radioactivity was also detected in a range of essential as well as non-essential amino acids.

Effects of maintaining Glossina morsitans morsitans on different hosts upon the vector's subsequent infection rates with pathogenic trypanosomes.

The percentage infection rates of Trypanosoma vivax in Glossina morsitans morsitans maintained after the infected meal on a cow, goats, rabbit, rats or mice were 88.0, 86.7, 94.8, 76.4 and 6.1, respectively. There were not significant differences between the males and females in this respect. The mortality rates of the tsetse maintained on mice or sheep were relatively high; the infection rate of the few survivors (5%) maintained on the latter host was 44.4%. The rates of T. congolense infection in the vector maintained on different hosts after the infected blood meal also differed. Goats (infection rate, 12.6%) and rabbit (11.4%) proved superior as maintenance hosts while rats (2.8%) and mice (0%) were inferior; sheep (7.8%) and cow (6.5%) were intermediate. Again, the mortality rate of the tsetse maintained on mice and sheep was markedly high; the reasons for this are discussed. Whereas cows (11.5%) and rabbit (11.2%) were efficient hosts for maintaining tsetse after the T. brucei infected feed, rats (4.1%) were inferior. Goat (9.0%) and mice (7.6%) proved intermediate. It is suggested that the rabbit is the best host for maintaining tsetse after infected feeds since the infection rates of all three Trypanosoma species in the vector were quite high, and this host is also relatively easy to handle in routine feeding of tsetse.

Studies on transmission of two East African stocks of Trypanosoma vivax to cattle, goats, rabbits, rats and mice.

Transmission studies were conducted using two Trypanosoma vivax stocks isolated from bovines in Uganda. Parasitaemia was low and transient in rabbits and rats; it persisted for relatively longer in NMRI mice. The parasitaemia developed to a peak in a few A/J and Balb/c mice; in NMRI, C57B and C3H/He it was low and fleeting. Lethally irradiated A/J, C57B and C3H/He mice with caesium 137 at 900 Gy showed a high peak of parasitaemia; NMRI and Balb/c mice succumbed very rapidly to a similar radiation dose. Serial maintenance of one stock of T. vivax was achieved in normal NMRI and lethally irradiated A/J mice. Both stocks failed to develop in the proboscis of Glossina morsitans morsitans or of G. m. centralis, and hence cyclical transmission to goats also failed. However, non-cyclical transmission by tsetse from goat to goat, and from cattle to goats, was successful. The infection caused acute and fatal disease in goats which were anaemic at death. The Boran cattle used eventually suppressed the infection and recovered.

Nutrition of Glossina morsitans: metabolism of U-14C threonine during pregnancy.

Following injection of U-14C threonine into the haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the postparturient female and in its offspring, in threonine, lipids, and a range of non-essential amino acids. The level of radioactivity recovered from the larva was higher than that remaining in the injected adult, and the radioactivity recovered was considerably higher in the amino acid than in the lipid fraction. Administration of labelled threonine into maternal haemolymph on each of the first 8 days of the 9-10 day long pregnancy cycle was followed 24 h later by measurement of radioactivity in the developing oöcyte and in the intra-uterine progeny. The patterns of nutrient uptake are discussed in relation to vitellogenesis in the oöcyte and to growth of the larva. Analysis of the expired carbon dioxide and excreta was carried out 24 h after maternal injection of labelled threonine on the first or eighth day of pregnancy. Carbon dioxide and excreta from females in early pregnancy showed significantly higher radioactivity than those from females in late pregnancy. In both cases, radioactivity in the amino acid fraction from the excreta was extremely small and about 95% of the total activity was in uric acid. These results are discussed in terms of the utilization of threonine in relation to the metabolic demands for various nutriments by the pregnant female.

New observations on cyclical development of Trypanosoma vivax in Glossina.

It is widely held that cyclical development of Trypanosoma vivax in Glossina is confined to the proboscis. This view has been re-examined in a series of experiments. Teneral G. morsitans centralis were fed on a goat infected with T. vivax IL 1392 and dissected 1-2 h after feeding. The infection rates in the labrum and hypopharynx were 40% and 0%, in contrast to 82% and 58%, respectively, observed in a control group dissected on day 25. This suggested that in a significant number of tsetse, cyclical development of T. vivax was initiated at sites other than the proboscis. Subsequent experiments revealed the presence of trypomastigotes, pre-epimastigotes and epimastigotes in the cibarium/oesophageal region of tsetse dissected 1-48 h after an infected feed. To investigate this further, tsetse proboscides were excised at intervals beginning 1 h after an infected feed, and transferred to in vitro culture conditions. Parasite multiplication and full cyclical development were only observed in proboscides excised 4 h or later after the infected bloodmeal. Thus, it would appear that at least in a number of tsetse, T. vivax cyclical development initially occurs in the cibarium/oesophageal region from where parasites migrate to the food canal of the proboscis and development is completed to infective metatrypanosomes in the hypopharynx.

Rickettsial infections of midgut cells are not associated with susceptibility of Glossina morsitans centralis to Trypanosoma congolense infection.

Teneral and 30-day old non-teneral Glossini morsitans centralis, from a laboratory-bred colony, were fed on a goat infected with Trypanosoma congolense clone IL 1180. They were then maintained on an uninfected rabbit, and dissected on day 30 after the infected feed. The midgut infection rates were 38.1% and 8.1%, with the mature infection rates of 28.7% and 4.3%, respectively. Electron microscopical examination revealed the presence of rickettsia-like organisms (RLOs) within the mycetomes and the midgut epithelial cells of all the teneral and non-teneral tsetse examined; the RLOs being more numerous in the older tsetse. Also, when the infected feed was given to teneral tsetse, maintained as above and dissected 30 days later, RLOs were observed in the tsetse with mature and immature T. congolense infections, as well as in those tsetse which completely lacked the trypanosomes. It appears that susceptibility of the laboratory reared G.m. centralis to T. congolense infection was not associated with RLOs within the midgut epithelial cells.

Suppression of cyclical development of Trypanosoma brucei brucei in Glossina morsitans centralis by an anti-procyclics monoclonal antibody.

Five hundred and sixty teneral male Glossina morsitans centralis were fed, at the height of parasitaemia, on a goat infected with Trypanosoma brucei brucei. Thereafter, the tsetse were divided into 4 equal groups. Group I was fed in vitro once weekly for 4 weeks and Group II twice weekly for 4 weeks on fresh defibrinated ox blood containing 2 mg/ml purified monoclonal antibody against T. b. brucei procyclics, while Group III was fed twice a week for 4 weeks on blood containing 2 mg/ml anti-T. vivax monoclonal antibody. The last group was fed on a rabbit. The tsetse were dissected on day 31 and the percent salivary gland infection rates observed were 18.2, 18.6, 39.8 and 40.8, respectively. In another experiment, 2 groups of tsetse, 120 per group, were fed on fresh defibrinated ox blood containing 2 mg/ml anti-T. b. brucei (test group) or anti-T. vivax (control group), on days 3, 6 and 9 following the infected feed. Dissection of the tsetse on day 31 revealed salivary gland infection rates of 0% in the test group and 6.5% in the control group. Thus the monoclonal antibody had a marked, specific suppression of the cyclical development of T. b. brucei in the tsetse vector.

The sequential cellular changes in the local skin reaction produced in goats by Glossina morsitans morsitans infected with Trypanosoma (Trypanozoon) brucei.

Sequential biopsies of the skin reaction elicited in goats by Glossina morsitans morsitans infected with Trypanosoma (Trypanozoon) brucei were examined histologically to identify and to quantify the cellular populations involved in the reaction. The peak of the tissue response occurred 7-8 days after challenge with infected tsetse and preceded the initial detection of parasitaemia by 4-5 days. Microscopically, the cellular reaction was characterized initially by a marked infiltration of polymorphonuclear leucocytes (PMN) which were replaced by lymphoid cells. Plasma cells and macrophages were numerous during the decline of the skin reaction. Marked enlargement of, and germinal centre formation in the regional lymph node accompanied the development and regression of the chancre. The results suggest that the formation of a chancre is dependent on the skin thickness of the host at the site of tsetse challenge.

The dynamics of the cellular reactions elicited in the skin of goats by Glossina morsitans morsitans infected with Trypanosoma (Nannomonas) congolense or T. (Duttonella) vivax.

Local skin reactions were elicited in goats by tsetse infected with either T. (N.) congolense of T. (D.) vivax. For the former trypanosomes, the skin reaction was detected initially 7 days after challenge and was maximal 3 days later. Histologically, the cellular response involved an initial influx of polymorphonuclear leucocytes (PMN) which was followed by a substantial infiltration of lymphocytes and macrophages. Large numbers of plasma cells remained in the skin reaction during its decline. Moderate numbers of parasites were observed in lesion at the height of the reaction. T. (D.) vivax provoked a small nodular skin reaction which became apparent 7 days after challenge. The cellular response, which peaked on day 9, contained large numbers of lymphocytes and macrophages and only a small contribution from PMN. Only small numbers of trypanosomes were observed in the chancre. The skin reaction elicited in goats by T. (N.) congolense, T. (D.) vivax or T. (T.) brucei were mutually distinct in their morphological appearance and size at the peak of the response, and in the interval required after challenge to attain maximum dimensions.

In vitro cultivation of animal-infective forms of a West African Trypanosoma vivax stock.

Animal-infective forms of a West African Trypanosoma vivax stock were grown in culture for three months using Minimum Essential Medium (MEM) with Earle's salts, supplemented with 20% inactivated goat serum over fibroblast-like cell lines isolated from the embryo of Microtus montanus or of an East African Galla crossbred goat at 36.5 degrees C and in 4% CO2 - 96% air. The bloodstream trypanosomes used to initiate the culture had been isolated from an infected goat. The cultured organisms grown in this system could be subcultured, were infective for mammalian hosts, retained their morphological characteristics and virulence, and could be readily established in Glossina morsitans centralis from goats injected with the cultured T. vivax.

Effects of carbon dioxide anaesthetic on Glossina.

Exposure of 2 day old virgin females of Glossina fuscipes fuscipes, G. pallidipes, G. brevipalpis, and G. morsitans centralis to carbon dioxide anaesthesia for 15 sec has the effect of suppressing their subsequent insemination frequency. This insemination-inhibitory effect of the gas is more pronounced in G. f. fuscipes and G. pallidipes than in G. brevipalpis and G. m. centralis. In G.f. fuscipes the adverse effect on insemination persists, albeit to a lesser degree, at least up to 72 hr. Carbon dioxide anaesthesia also reduces the insemination capability of G.f. fuscipes males; this effect, however, is less marked than in females. Exposure of wild non-teneral G.f. fuscipes, G. pallidipes, G. brevipalpis, and 3 day old G.m. centralis to the gas for 30 min causes some mortality during anaesthesia, which increases with increasing exposure period. G. m. centralis is most tolerant to the lethal effect of the gas within the 10-90 min exposure periods. Wild females of G.f. fuscipes and G. pallidipes seem to be more sensitive to carbon dioxide in this report than males. In view of these adverse effects produced by carbon dioxide anaesthesia, its use on tsetse is not to be recommended.

Expression of resistance to isometamidium and diminazene in Trypanosoma congolense in Boran cattle infected by Glossina morsitans centralis.

Investigations were conducted on the sensitivity to isometamidium chloride (Samorin) and diminazene aceturate (Berenil) of derivatives of three of the Trypanosoma congolense stocks isolated between 1978 and 1983 from Zebu cattle in the Bobo-Dioulasso region of Burkina Faso. Boran cattle were used in the drug-sensitivity tests and were infected using Glossina morsitans centralis. The results showed that T. congolense stock IL 2466 isolated in 1978 was sensitive to the standard therapeutic dose of isometamidium chloride (0.25 mg kg-1) and of diminazene aceturate (a.i. 3.5 mg kg-1). However, T. congolense stock IL 2468 isolated in 1982 was resistant to both the prophylactic (0.5 and 1.0 mg kg-1) as well as the therapeutic doses of isometamidium chloride (up to 1.0 mg kg-1) although the sensitivity to the therapeutic dose of diminazene aceturate (3.5 mg kg-1) was not affected. The T. congolense stock IL 2856 isolated in 1983 was highly resistant to the therapeutic action of diminazene aceturate (up to 10.5 mg kg-1), as well as to the prophylactic (up to 1.0 mg kg-1) and therapeutic action of isometamidium chloride (up to 2.0 mg kg-1). The infection rates of the drug-resistant stocks of T. congolense in G.m. centralis, when goats were used as reservoir hosts, were as high (range, 22.3-56.3%) as of the drug sensitive stock (49.5%). The resistance trait in the two stocks remained stable after their cyclical development in the tsetse vectors. The rate of transmission of the drug-resistant stocks to mice by the infected tsetse was also high (mean 81.3%).

Trypanosoma simiae: in vitro studies on drug susceptibility.

Two Trypanosoma simiae stocks were initiated in culture with tsetse-derived metacyclics. They were propagated axenically as trypomastigote forms at 35 degrees C in 4% CO2 in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. Diminazene aceturate, quinapyramine sulphate, DL-alpha-difluoromethylornithine, and Ro 15-0216 showed activity against the stocks. Suramin and Mel Cy showed little effect upon the growth of the parasite populations. Isometamidium chloride gave questionable results in the 24 h growth inhibition test, but the results of a long-term viability assay indicated some degree of drug resistance (or drug tolerance). The results obtained herein correlate with observations obtained from in vivo studies in pigs. It is thus concluded that in many cases the cryptic nature of T. simiae rather than drug resistance is responsible for the failure of chemotherapy of simiae-trypanosomiasis in pigs.

Infection rates in sterile males of morsitans, palpalis and fusca groups Glossina for pathogenic Trypanosoma species from East and West Africa.

Infection rates in sterile male Glossina morsitans centralis, G. austeni, G. palpalis palpalis, G.p. gambiensis, G. fuscipes fuscipes, G. tachinoides and G. brevipalpis for Trypanosoma vivax, T. congolense and T. brucei isolated from East and West Africa, were studied. Five groups of the sterile males, together with the five groups of sexually fertile males, of each of the respective species and subspecies were allowed to feed for 24 days on a Boran calf or goats infected with T. vivax or T. congolense, or with T. brucei for 34 days, after which they were dissected. The results showed that the infection of the pathogenic Trypanosoma species became better established in some tsetse species than in others. Also, the infection rates of T. vivax, T. congolense and T. brucei for sterile and sexually fertile males of any of the above Glossina did not differ significantly. These results indicate that releases of sterile male tsetse in the tsetse control programme will potentially increase the risk of trypanosomiasis during the period of tsetse releases in the affected areas, unless in the areas with low tsetse density the sterile male tsetse are rendered refractory to trypanosome infection prior to their releases while in the areas with medium to high tsetse densities, the resident tsetse populations are initially reduced with insecticides, traps and/or targets.

Failure of trypanosomal membrane antigens to induce protection against tsetse-transmitted Trypanosoma vivax or T. brucei in goats and rabbits.

A purified protein, relative molecular weight 83 kilodalton (kD), and plasma membranes from Trypanosoma brucei were tested as potential vaccines against tsetse-transmitted T. vivax and T. brucei in goats and rabbits. The 83 kD protein was found in lysates of all clones of T. brucei examined, as well as in lysates of T. vivax, T. congolense and T. rhodesiense. Rabbits and goats were immunized with various amounts of antigen in Freund's complete adjuvant and boosted twice with antigen in Freund's incomplete adjuvant. Two weeks after the last inoculation, the goats were challenged with T. vivax-infected and the rabbits with T. brucei-infected Glossina morsitans morsitans. Although high antibody levels were detected in all the animals immunized with either antigen as measured by radioimmunoassay and immunodiffusion, they became infected and the course of disease was the same as that in unimmunized controls.

Parasite kinetics and cellular responses in goats infected and superinfected with Trypanosoma congolense transmitted by Glossina morsitans centralis.

Trypanosoma congolense infected tsetse were fed on the flanks of goats at sites drained by the prefemoral lymph node. The efferent lymphatic of this lymph node was surgically cannulated and the lymph was collected daily and examined for appearance of parasites, lymph flow and cells. Trypanosomes were detected in the lymph 4 days after infection, which was 2 days prior to the appearance of the local skin reaction or the presence of parasites in the blood. Once the animal became parasitaemic, trypanosomes were found to recirculate in the lymphatic system, appearing in the lymph of the contralateral lymph node 11 days after infection. In goats infected with T. congolense and superinfected 12 or 13 days later with a different tsetse-transmitted T. congolense serodeme, parasites belonging to the second serodeme were apparently delayed in their development in the skin and appeared up to 7 days later in the efferent lymph when compared to control animals. This delay in development might have implications for field situations where superinfections frequently occur; it might result in limiting the number of serodemes of T. congolense an animal can be infected with at any one time.