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SYNOPSIS An account is given of uric acid accumulation in the egg of Schistocerca gregaria during the course of embryonic development and its storage sites in the egg.

Two colonies of Glossina pallidipes originating from the Shimba Hills and Nguruman, Kenya, were examined for their susceptibility to two strains of Trypanosoma congolense and for allele frequencies at eight polymorphic loci. These data were compared with data from earlier studies of these colonies. Overall, the two colonies of G. pallidipes were found to have lost, over 3-4 years, most of the genetic and trypanosome-susceptibility characteristics that originally distinguished them.

In a single generation of selection, two lines of Glossina morsitans centralis were established that differed significantly in susceptibility to Trypanosoma congolense clone IL 1180. Reciprocal crosses demonstrated that susceptibility was a maternally inherited trait. Differences between the lines, to all phases of the trypanosome infection, were maintained for eight generations, whereas differences in susceptibility to midgut infections were maintained for twenty-eight generations. Thereafter, the lines did not differ in susceptibility to Trypanosoma congolense IL 1180. Susceptibility to infections with Trypanosoma congolense IL 1180 was only a weak predictor of susceptibility to T. congolense clones IL 13-E3 and K60/1, as well as clone T. brucei brucei STIB 247-L. However, the susceptible and refractory lines displayed these phenotypes when tested with Trypanosoma vivax, indicating that the factors that affect susceptibility to trypanosomes are expressed both within and outside the midgut.

Two laboratory strains of Glossina morsitans centralis originating from different fly-belts (one from Singida, in Tanzania, and the other from Mumbwa, in Zambia) were compared with respect to vectorial competence for pathogenic Trypanosoma species, genetic variation and inter-colony fertility. The vectorial competence of G.m.centralis of Tanzanian origin for Trypanosma vivax and T. congolense is similar to, whereas for T.brucei brucei it is lower than the colony of Zambian origin. Nevertheless, these two laboratory strains of G.m.centralis showed levels of susceptibility to the three pathogenic Trypanosoma species which were much greater than previously observed in laboratory colonies of other Glossina species. Electrophoresis of fifteen enzymes revealed that the two colonies differ significantly in allele frequencies at only three loci that are relatively close together on one of the autosomes. Hybridization experiments revealed that G.m.centralis from the two fly-belts are consubspecific.

A colony of Glossina pallidipes which originated from Nguruman, Rift Valley Province, Kenya, was significantly more susceptible than a colony of the same species which originated from Shimba Hills, Coast Province, Kenya, to infection with a stock of Trypanosoma simiae CP 11 isolated from wild G. austeni in Coast Province, Kenya, irrespective of whether pigs or goats were used as infecting hosts. Male G. pallidipes from both the colonies were more susceptible than females to this T. simiae stock. Similarly, a G. pallidipes colony of Nguruman origin was significantly more susceptible than the colony of Shimba Hills origin to infection with another stock of T. simiae CP 813 isolated from wild G. pallidipes in Coast Province, Kenya, again irrespective of whether pigs or goats were used as infecting hosts. The susceptibility of the sexes of G. pallidipes from both the colonies to T. simiae CP 813 did not differ significantly when pigs were used as infecting hosts, but male G. pallidipes from both the colonies were significantly more susceptible than female tsetse to this T. simiae stock when goats were used as infecting hosts. Nevertheless, if the observed differences in susceptibility of the two G. pallidipes colonies reflect transmission of trypanosomes by the two allopatric populations of tsetse in the field, then the epidemiology of simiae-trypanosomiasis probably differs between these two areas of Kenya.

A colony of Glossina pallidipes Austen which originated from Nguruman, Rift Valley Province, Kenya, was significantly more susceptible to infection (19.3%) with a stock of Trypanosoma congolense Broden isolated from G. pallidipes in Nguruman than a colony of the same species which originated from Shimba Hills, Coast Province, Kenya (5.6%). Male G.pallidipes from Nguruman were significantly more susceptible than females to this T.congolense stock whilst the susceptibility of both sexes of G.pallidipes from Shimba Hills did not differ significantly. All six goats on which six infected G.pallidipes fed singly (three tsetse per colony) became infected. Similarly, the G.pallidipes colony of Nguruman origin was significantly more susceptible to infection (16.4%) with a stock of T.congolense isolated from G.pallidipes in Shimba Hills than the colony of Shimba Hills origin (4.9%). The susceptibility of the sexes of G.pallidipes from both the colonies to this stock of T.congolense did not differ significantly. Again, all six goats on which six infected G.pallidipes fed singly (three tsetse per colony) became infected. If the observed differences in susceptibility of the two G.pallidipes colonies reflect transmission of trypanosomes by the two allopatric populations of tsetse in the field, then the epidemiology of congolense-trypanosomiasis in livestock must differ between these two areas of Kenya endemic for trypanosomiasis.

Twenty-five goats were randomly allocated to five groups of five animals each and infected with Trypanosoma congolense IL 3274 via the bites of infected Glossina morsitans centralis. At intervals of 1, 4, 8, 12 or 19 days following infection, each group of five animals was treated intramuscularly with diminazene aceturate at a dose of 7.0 mg kg-1 body weight (b.w.). While treatment on Day 1 eliminated infections in all five goats, treatment on Day 19 did not cure any of the animals; in groups treated 4, 8 or 12 days following infection, two of five goats in each group were cured. Since the alteration in apparent resistance of T. congolense IL 3274 between Day 1 and Day 19 could have been due to alteration in expression of drug resistance by trypanosomes as the population expanded, the experiment was repeated using trypanosomes that reappeared in the animals that had been treated with diminazene aceturate on Day 19. On Day 36, when all five animals were parasitaemic, five groups of teneral G. m. centralis, each containing 160 flies, were fed on one occasion on each of the five goats (one group of testse flies per goat). Thereafter, each group of tsetse flies was maintained on clean rabbits. When infective, five flies from each group were allowed to feed on two naive goats each (i.e. two goats per group of tsetse flies). One animal in each pair was treated 24 h after infection with diminazene aceturate at a dose of 7.0 mg kg-1 b.w., the other was treated on Day 19, when parasitaemic, with the same drug dosage. As before, treatment 24 h following infection eliminated infections in all animals, but when treatment was delayed until Day 19, trypanosomes in all animals were refractory to treatment. Thus, although tsetse flies were infected with trypanosomes that had arisen in infected goats following treatment with diminazene aceturate at a dose of 7.0 mg kg-1 b.w., when the same flies were allowed to feed on clean goats, the resultant infections were sensitive to treatment with the same drug dosage when administered 24 h following infection. These data therefore indicate that there is a significant alteration in diminazene sensitivity of IL 3274 between Day 1 and Day 19 and that this is associated with an alteration in the resistance phenotype of the trypanosomes.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.

SUMMARYThe midgut epithelium ofGlossina morsitans centralis, G. austeni, G. pallidipes, G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes, G. tachinoidesandG. brevipalpisfrom ILRAD-bred colonies was examined, by electron microscopy, for the presence and distribution of Rickettsia-like organisms (RLOs). RLOs were present in the midgut epithelial cells of all non-teneral tsetse. InG. m. centralis, G. pallidipesand, to a much lesser extent,G. brevipalpis, RLOs were numerous and were present in all the specimens examined. RLOs were present in fewer numbers in the epithelial cells of tenerals of these three tsetse species. In contrast, RLOs occurred in very much lower numbers within the midgut cells of nonteneralG. austeni, G. p. palpalis, G. p. gambiensis, G. f. fuscipesandG. tachinoides; were not seen in every specimen, and were rarely observed in the midgut cells of teneral tsetse. The RLOs were typical rod-shaped bacteria with an inner and outer membrane, which occurred free within the host cell cytoplasm and appeared to cause no obvious pathology. The micro-organisms divided by binary fission and at least two distinct morphological forms plus a range of intermediate forms were seen in the midgut cells. A comparison of the presence and numbers of RLOs within the midgut cells and the midgut infection rates of bothTrypanosoma congolenseandT. b. brucei, both betweenGlossinaspecies and also within the same stock of tsetse, clearly indicates that the ability of trypanosomes to establish and develop to mature infections is unlikely to be correlated solely with the presence of RLOs within the tsetse midgut.

Investigations were conducted on the sensitivity to isometamidium chloride (Samorin R ) and diminazene aceturate (Berenil R ) 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 theapeutic 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%).

(1992). Virulence of Trypanosoma simiae in pigs infected by Glossina brevipalpis, G. pallidipes or G. morsitans centralis. Annals of Tropical Medicine & Parasitology: Vol. 86, No. 6, pp. 681-683.

Teneral tsetse of four Glossina species from laboratory-reared colonies were fed on four Large White pigs infected with three different stocks of Trypanosoma simiae isolated in Coast Province, Kenya. Thereafter the tsetse were maintained on goats and dissected on day 28 to determine the trypanosome infection rates. Glossina brevipalpis was as susceptible as G.pallidipes whilst G.palpalis gambiensis was not susceptible to T.simiae CP 11 a stock causing acute infection, which was isolated from a wild G.austeni. Glossina brevipalpis was as susceptible as G.pallidipes to another stock causing acute infection, T.simiae CP 813 isolated from a wild G.pallidipes. Glossina morsitans centralis was also as susceptible as G.brevipalpis and G.pallidipes whilst G.p.gambiensis was not susceptible to this T.simiae stock. Glossina m.centralis showed very low susceptibility to a stock causing chronic infection, T.simiae CP 1896 isolated from a bushpig, whilst G.brevipalpis, G.p.gambiensis and G.pallidipes could not be infected by this T.simiae stock. Male Glossina were generally more susceptible than females to the three T.simiae stocks.

Two Trypanosoma vivax stocks were initiated in culture with tsetse or culture-derived metacyclics. They were propagated axenically as bloodstream trypomastigotes at 35 degrees C in 4% CO2 in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds, namely diminazene aceturate, quinapyramine sulphate, DL-alpha-difluoromethylornithine, isometamidium chloride, suramin and melCy. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. All six drugs tested showed little effect upon the growth of the parasite populations. These results indicate that a 24-h growth-inhibition test was not suitable for determining the drug susceptibility of T. vivax stocks in vitro. Neither did the results correlate with those obtained with susceptible or resistant stocks of T.b. brucei, T.b. evansi or T. simiae described in the literature, or with the results of these two T. vivax stocks tested in cattle.

Two large colonies, originating from allopatric populations of Glossina pallidipes Austen, in the Shimba Hills and Nguruman, Kenya, which differ biologically and with respect to vectorial competence, were compared at fourteen enzyme loci using polyacrylamide gel electrophoresis. The colonies had similar levels of genetic diversity with approximately half of the loci being polymorphic, an average of 1.6-1.7 alleles per locus, and a mean heterozygosity per locus of approximately 18.4%. However, the colonies differed significantly in allele frequencies at the loci for phosphoglucomutase, glucose-6-phosphate dehydrogenase, xanthine oxidase, octanol dehydrogenase and phosphoglucose isomerase. The results were compared with earlier studies on this species and no evidence was found for selection of specific alleles during establishment or maintenance of colonies of G. pallidipes, nor were specific chromosomes, or marker genes, associated with the biological differences between the two colonies.

A black rhinoceros (Diceros bicornis) moved from a tsetse-free to a tsetse-infested area in Kenya was monitored for two months following translocation. The animal acquired a Trypanosoma vivax infection from natural tsetse challenge, but survived without requiring treatment with trypanocides. The infection was characterised by moderately high parasitaemia, with symptoms of anaemia, leukopaenia and thrombocytopaenia. Although confirmed to be T. vivax through deoxyribonucleic acid hybridisation and parasite development in tsetse in the proboscis only, the parasite had unusual morphology and motility. It also failed to infect normally susceptible hosts such as cows and goats, and produced unusually low infection rates in Glossina morsitans centralis and G. brevipalpis.

Diminazene aceturate is one of a limited number of compounds currently marketed for treatment of trypanosomiasis in cattle, sheep and goats. The pharmacokinetics of the compound in goats suggest that double treatment with diminazene aceturate might enhance the compound's therapeutic activity. A study was therefore conducted in goats using two clones of Trypanosoma congolense, IL 3274 and IL 1180, which were previously shown to be resistant and sensitive, respectively, to single treatment with diminazene aceturate. The results indicated that, as compared to single treatment, double treatment with diminazene aceturate at a dose of 7.2 mg kg-1 bodyweight, at either eight or 24 hour intervals, did not greatly enhance the therapeutic activity of the drug. Furthermore, treatment with the same drug dose eliminated infections with T congolense IL 3274 when treatment was administered 24 hours after infected Glossina morsitans centralis had fed, but failed to do so if treatment was delayed until after goats were detected to be parasitaemic. This suggests that failure of T congolense IL 3274 to respond to treatment with diminazene may not be due to drug resistance per se.

Glossina brevipalpis Newstead, G.longipennis Corti, and G.pallidipes Austen maintained at ILRAD, Nairobi, Kenya, were examined for genetic variation of fourteen enzyme loci, using polyacrylamide gel electrophoresis. G.brevipalpis had six polymorphic loci, an average of 1.46 effective alleles per locus and a mean heterozygosity per locus of 20.0 +/- 7.1%. The figures for the same parameters in G.longipennis were 3, 1.16 and 8.2 +/- 4.9%, and for G.pallidipes the figures were 7, 1.40 and 22.3 +/- 6.3%. Seven rare alleles were lost from the G.brevipalpis colony during a 1-year period, but no statistically significant changes were observed in the genetics of the colony during this period. Using allele frequency data for ten of the enzymes studied, and frequencies for these enzymes in other taxa, a phenogram was constructed that indicated that the subgenus Austenina (i.e. the fusca group) is the oldest of the three subgenera within the genus Glossina, and that the subgenus Glossina s.str. (i.e. the morsitans group) may be paraphyletic.

Feeding behaviour of Glossina palpalis gambiensis Vanderplank infected with Trypanosoma vivax Ziemann was studied and compared with that of uninfected control tsetse. The parameters measured were: total number of probes into the ear-skin of rabbits; rate of bloodmeal engorgement; weight of freshly ingested blood; survival; and mean weight of pupae. The results showed that the rosettes of T.vivax parasites in the labrum did not interfere with the feeding behaviour of the vectors. Furthermore, mean survival of T. vivax-infected males was significantly higher (82.2 +/- 4.2 days) compared with that of uninfected ones (70.5 +/- 3.1 days). However, with the female tsetse, mean survival of those infected was lower (98.8 +/- 4.0 days) compared to the uninfected controls (102.2 +/- 5.6 days), but the difference was not significant. A few infected males and females lived a little longer than the uninfected ones. Fecundity of the female tsetse remained unaffected by the infection, and furthermore the mean weight of pupae from the infected females was not significantly different from that of pupae from the uninfected control group. Thus the physiology of pregnant female tsetse in terms of nourishment of intra-uterine larva was unaffected by T.vivax infection. Two successive probes into the skin of two different goats followed by feeding on a third goat by each of four infected tsetse resulted in successful transmission of the infection to eleven out of twelve goats. Thus probing alone into the skin of this host can result in the transmission of T.vivax infection.

Under simulated natural conditions the anal orifice of the larva of Glossina morsitans morsitans Westw. appears to open either at or soon after parturition. The larva burrows on average within two minutes of deposition; pupariation is completed 10–15 min later. Using methods that prevent burrowing and prolong the crawling period, certain larval movements were related to the pre-pupariation anal discharges and to the rapid spread of the fluid. Although the anus is not generally thought to be connected with the hind gut during the whole of larval life, the presence of uric acid in the discharges indicates that the fluid is a product of Malpighian tubule excretion. In a two-choice chamber in an incubator, flies larviposited significantly more often in the experimental pot if it contained a water- or ether-soluble extract of larval excretion in addition to any pupariating larvae. This points to the presence of a larval phero-mone which may attract gravid females to a breeding site.

The infection rates and transmission characteristics of sterile male Glossina morsitans centralis, G. austeni and G. tachinoides for Trypanosoma vivax, T. congolense and T. brucei were studied. Teneral males were irradiated with 10 krad in a 137caesium source under ambient conditions. Five groups of teneral males of each species were sterilised by irradiation with 10 krad caesium source under ambient conditions and then fed for 3 days, together with five groups of normal males of their respective species, on three goats infected with T. vivax, T. congolense or T. brucei. The mean sterility rates were 98, 93 and 90% for G. m. cenlralis, G. austeni and G. tachinoides respectively. The mean percentage mature infection rates in the irradiated as against the controls of G. m. centralis, G. austeni and G. tachinoides were respectively 27.3 ± 9.5/27.7 ± 11.1, 4.0 ± 1.4/4.0 ± 2.6 and 14.8 ± 7.0/12.2 ± 5.0 for T. vivax; 24.6 ± 5.2/30.3 ± 4.3, 0.7/0.3 and 0.3/0.3 for T. congolense; and, 22.7 ± 2.6/23.4 ± 3.1, 1.1/0.2 and 0.7/1.7 for T. brucei. The transmission rates and frequency to A/J mice were very high and did not differ between the sterile and normal males. There was also no difference in the pattern of the diseases in the goats challenged by sterile or sexually fertile males of the three tsetse species. These results indicate that releases of sterile male tsetse in tsetse control programmes will potentially increase the trypanosomiasis risk in the affected area. Hence appropriate measures should be integrated in any SIR method of tsetse control campaign in order to provide adequate protection to man and his associated livestock.

Rabbits exposed to feeding tsetse flies developed cutaneous hypersensitivity responses to fly bites. These responses had characteristics of immediate and delayed type hypersensitivity. Saliva components from the tsetse fly Glossina morsitans centralis were electrophoretically separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Major salivary proteins of 160, 92, 66, 64, 55, 42, 33, 28, and 15 kilodaltons were identified. Separated salivary components were transferred to nitrocellulose filters and probed with lectins and with whole sera and purified IgG from rabbits which had been exposed, via fly feeding, to tsetse antigens for variable periods. Many of the salivary proteins were identified as glycoproteins. Several major salivary proteins were recognized by antibodies from sensitized rabbits.

SUMMARYCoated metacyclics ofTrypanosoma vivaxexist in the hypopharynges of infected tsetse flies and are extruded in low numbers when the flies are induced to probe onto warm slides or into medium. After extensive searching ofT. vivax-infected proboscides, and resort to a process for the examination of single, extruded, metacyclic trypanosomes, electron micro scopic evidence is presented that, contrary to an earlier report, metacyclicT. vivaxacquire a surface coat before contact with the mammalian host. SinceT. vivaxexhibits antigenic variation, the role of the surface coat in this species is likely to be functionally equivalent to the surface coat of the other tsetse-transmitted trypanosome species,T. bruceiandT. congolense.

In a follow-up survey of sleeping sickness in the Ikoma-Serengeti region of Tanzania, carried out in May and June 1972, 11,060 G. swynnertoni and 95 G. pallidipes were collected, triturated mainly in batches of 50, and the supernate of each batch was inoculated into cyclophosphamide-treated mice. Nine strains of brucei-subgroup were isolated from G. swynnertoni, which indicated the vector ro1e of the local tsetses in the transmission cycle of infection in this endemic region. All the 9 brucei-subgroup isolates gave consistently negative results by the Blood Incubation Infectivity Test (BIIT) whereas the respective controls gave positive results. However, T. (T.) brucei and particularly T. (T.) rhodesiense standards which were tested simultaneously with the test strains gave inconsistent results. It would seem therefore that the BII test in its present form is not reliable for differentiating between the above two trypanosome species, and so the 9 brucei-subgroup isolates could not be classified further. In the Ikoma-Serengeti region there are no restricted foci of Rhodesian sleeping sickness but rather the various components comprising zoonosis interact sporadically throughout this part of Tanzania. The biocenosis of infection favours intense circulation of the parasites, mainly via the tsetse vectors, among game animals some of which function as natural reservoirs; in this ecological zone man is only an incidental host.

SUMMARYInvestigations were conducted on the infection rates and transmission charecteristics of sterile male Glossina morsitans morsitans for Trypanosoma vivax, T. congolense and T. brucei. Pupae were irradiated after the first flush of female emergences with 7, 10 or 13 krad. in a 137caesium radiation source under ambient conditions. The emergent males were fed on a T. viuax-infected goat at peak parasitaemia. They were subsequently mated at 8 days old with 3-day-old normal, virgin females. A batch of sexually fertile males which had been infected as tenerals on the goat at the same time were similarly mated to serve as controls. All the tsetse were maintained on rabbits' ears; the survival and reproductive performance were monitored for seven age-group periods of 9 days each. The sterility rates of the males were 73, 91 and 98% for the 7, 10 and 13 krad. doses of radiation; the percentage of T. vivax infection rates were 78·3, 82·3 and 74·4, respectively. For the control males, the fecundity of the mated females was 90% and the infection rate was 75·7%. In all other experiments, the 10 krad. dose was selected since both the survival and the sterility of the irradiated males were good. The T. vivax-challenged cows and goats died with symptoms of anaemia. There was no difference in the transmission characteristics between sterile and fertile vectors. There was also no significant difference between the sterile and normal males in the infection rates and the transmission frequency of T. congolense and T. brucei to cattle, goats and mice. These results indicate that release of sterile male tsetse in tsetse control programmes will potentially increase the trypanosomiasis risk in the affected area. Consideration must therefore be given to integrating adequate surveillance and chemotherapeutic measures for protection of man and his domestic livestock in the region during the period of such tsetse control campaigns.

Glossina pallidipes, G. f. fuscipes and G. brevipalpis in Stages I and II of hunger were caught from Bukunya and Buyundo villages in south-east Uganda during the period December 1969 to February 1971, and the origins of their blood meals identified. G. pallidipes fed mainly on bovids in the two villages; bushbuck was the most favoured host (44.9% of all blood feeds) though in Buyundo buffalo provided a significant alternative (20.8%). G. f. fuscipes frequently fed on bovids (75.6%), particularly bushbuck (46.7%), but it had a wider range of acceptable hosts. In both villages, G. brevipalpis took most of its blood meals from bushpig (45.5%), bovids (25.6%) and hippopotamus (28.5%). The feeding patterns of the three tsetse species during different months of the study period showed only small variations, and such differences were due probably to variations in the availability of the hosts. Examination of the relationships between trypanosome infection rates and natural hosts of the three vector species showed that the vivax-type infections originated from bovids, possibly bushbuck, while the congolense-type trypanosome came from bovids as well as bushpig. Hippopotamus probably had no trypanosome infections. There was a positive correlation between the trypanosome infection rates in the tsetse and the increasing mean maximum temperatures. In this region of Uganda, there was continuous transmission of pathogenic trypanosomes by the three tsetse species among their natural hosts; when present, the vectors' adventitious hosts became exposed to a high risk from the diseases. Man-fly contact during different months was impersonal, but high and sporadic. This is a high endemic region for human trypanosomiasis. Also, the introduced cattle succumb to the diseases very rapidly. The overall results are discussed in terms of the concept of challenge of African trypanosomiases.

Twenty-four Boran cattle were injected with isometamidium chloride (1 mg/kg bodyweight) to investigate the duration of drug-induced prophylaxis against infection by metacyclic forms of Trypanosoma congolense and to determine if specific antibody responses to the organism were mounted by animals under chemoprophylactic cover. Complete protection against either single challenge by five tsetse flies infected with T congolense, or repeated challenge at monthly intervals by five tsetse flies, lasted for five months. Six months after treatment, two-thirds of the cattle were resistant to challenge, irrespective of whether subjected to single or multiple challenge with trypanosome-infected tsetse flies, or titrated doses of in vitro-cultured metacyclic forms of T congolense (5 X 10(2) to 5 X 10(5) organisms), inoculated intradermally. No animal which resisted infection developed detectable skin reactions at the site of deposition of metacyclic trypanosomes or produced trypanosome-specific antibodies. It was concluded that drug residues effectively limited trypanosome multiplication at the site of deposition in the skin, thus preventing subsequent parasitaemia or priming of the host's immune response.

Investigations were conducted on the infection rates of a West African stock of Trypanosoma vivax in Glossina morsitans morsitans and G. m. centralis. The mature infection rate, as revealed by the warm-slide probe, was only 9·4% in contrast to 67·7% by the dissection method. The infection rates determined by the latter technique varied between 46·;7 and 100%, and there were no significant differences in the rates between tenerals and non-tenerals, males and females, the two sub-species, the different age of the vectors or the different peaks of parasitaemia in the goats at the time of the infective meal. In some tsetse the number of parasites in the hypopharynx was markedly high, and there was no significant difference in the frequency of such vectors between the sexes. The mean infection rates in G. m. morsitans increased with increase of the parasitaemia in the host goats. The overall results are discussed in terms of the field situation.

After injections of U-14C-phenylalanine into the haemolymph of female Glossina morsitans Westw. during late pregnancy, radioactivity was detected in the post-parturient female and its larval offspring in the injected material and in lipids, a range of non-essential amino acids and tyrosine. After intrahaemocoelic administration of U-14C-tyrosine, radio-activity was detected in the adult and larva in all these nutriments except phenylalanine. The level of radioactivity in the larva was higher than that remaining in the female parent, and in both radioactivity was greater in amino acids than in the lipid fraction. Radiometric analysis of the oocyte and the infra-uterine larva 24 h after injection of labelled phenylalanine or tyrosine on different days of the pregnancy cycle revealed patterns of radioactivity relative to their respective growth characteristics. The rate of phenylalanine utilisation by the pregnant female for nourishment of the in-utero third-instar larva was significantly higher than the rate of tyrosine utilisation. For both labelled materials, there was a significantly higher utilisation for energy during early than during late pregnancy, but at both physiological ages tyrosine was metabolised for energy to a relatively greater degree. Uric acid is the main nitrogenous waste product of aromatic amino acid metabolism in this insect, though a very low level of activity was also detected in the amino acid fractions of the excreta from females in early and late pregnancy.

The degree of determination of the young embryo of S. gregaria has been investigated using ligation, thermocautery and centrifugation techniques. From the overall results, it is suggested that the early development of the embryo is mediated by two physiological centres. The formation of the germ rudiment is controlled by an activation centre located in the periplasm round the posterior end of the egg. This centre is already present at the zygote nucleus stage and is essential during the very early cleavage period. The differentiation of the germ band is induced by the activity of a second centre, the differentiation centre, located in the presumptive thorax. It apparently becomes established at least by the late blastoderm stage and its activity continues during the period of germ-band formation. During the late cleavage and early blastoderm stages, the egg is labile and the embryo is therefore able to normalize its development after part or parts of the germinal Anlage have been cauterized, removed or displaced. The differentiation centre completes its functions by the beginning of gastrulation. Thereafter, the embryo is determined. The embryo can regulate its size at least up to the gastrulation stage provided that a certain minimum amount of usable yolk is available. The development of the serosa is not under the control of either centre. This structure seems to be capable of regeneration providing that a part of the extra-embryonic blastoderm remains intact.

Records were kept of the catches of tsetse fly by three types of traps, the Langridge box screen, the Swynnerton awning screen, and a modification of the latter incorporating a screen skirt, in the South Busoga fly-belt, Uganda, where Glossina pallidipes Aust., G. fuscipes Newst. and G. brevipalpis Newst. are abundant. Only two specimens of G. brevipalpis, but large numbers of the other two species, were caught, by far the largest catches being obtained by the awning screen-skirt trap.

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.

A study of the epidemiology of animal trypanosomiasis was carried out on a 2500 ha cattle ranch, with a history of trypanosomiasis, in the Coast Province of Kenya in 1982. The tsetse survey on the ranch revealed one breeding focus of Glossina austeni in a thicket of approximately 50 ha. Trypanosomes were detected in 20% of the 46 dissected tsetse. During the study period of 9 months, 0.8% of the 3315 samples collected from 2300 Ayrshire X Sahiwal crossbred cattle were found infected with trypanosomes; 32% of 5909 samples collected from the same cattle had a packed cell volume (PCV) of 30% or less. Animals with a PCV of 30% or less were treated with a trypanocide (Berenil, Ethidium or Novidium). Antibody to trypanosomes was detected in 22.1% of the 343 sera collected from the cattle. A sentinel herd of 20 cattle was exposed for 182 days inside the tsetse infested thicket. All animals became infected with Trypanosoma congolense, on average after 53 days; they were subsequently treated with Berenil (6 mg/kg). A second, third and fourth T. congolense infection was diagnosed in 17, 11 and 1 animals, respectively. The cattle were treated similarly with Berenil after each of these infections. T. vivax and T. brucei were not diagnosed in the sentinel cattle. The results suggest that acquired immunity to T. congolense infection did not play a significant role in the sentinel cattle.

SUMMARYTeneral maleOlossina morsitans centralis, G. austeni, G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes, G. tachinoidesandG. brevipalpiswere fed on the flanks of Boran calves infected withTrypanosoma vivaxstock ILRAD 2241 isolated from a cow in Likoni, Kenya; stock ILRAD 2337 isolated from a cow in Galana, Kenya; stock ILRAD 1392 isolated from a cow in Nigeria; or, stock EATRO 1721 isolated fromG. m. submorsitansin Nigeria. The tsetse were fed on the infected hosts for 24 days and were then dissected to determine the infection rates. InG. m. centralisandG. brevipalpis, the mature infection rates ofT. vivaxfrom Kenya were 61·1 %, and 75·3% for ILRAD 2241, and 36·2% and 58·2% for ILRAD 2337, respectively. InG. austeniand in the fourpalpalisgroup of tsetse, the rates for these two stocks were very low and ranged from 0% inG. p. palpalisto 1·8% inG. austenifor ILRAD 2241 and from 0% inG. f. fuscipesto 5% inG. tachinoidesfor ILRAD 2337. In contrast, the hypopharyngeal infection rates ofT. vivaxfrom Nigeria were quite high in all the 7 tsetse species and sub-species. They ranged from 55·5% inG. austenito 919% inG. p. gambiensisfor ILRAD 1392, and from 71·4% inG. austeneito 97·1 % inG. brevipalpisfor EATRO 1721. It is suggested that successful establishment ofT. vivaxinfection in a particular tsetse species could depend on the biochemical characteristics of its attachment sites in the food canal and the efficiency of bloodstream trypomastigotes of a particularT. vivaxstock to attach to such sites and undergo complete development to meta-trypanosomes in the hypopharynx of the vector.

The development and distribution of Trypanosoma congolense, T vivax and T brucei in the skin of goats was examined after the animals were bitten by infected Glossina morsitans centralis. Following the tsetse bite, the trypanosomes in the skin multiplied, reaching maximum numbers when the skin reaction (chancre) of the host attained its maximum size. In goats infected with T vivax and T brucei, trypanosomes were observed circulating in the blood before the peak of the chancre, while in T congolense-infected goats microscopically detectable parasites were found in blood only during the decline of the chancre. In contrast to T vivax, large numbers of T congolense and T brucei parasites were found in the skin following tsetse-transmitted infection. Ultrastructural differences were observed in T congolense and T brucei indicating an intracutaneous transformation from metacyclic to blood stream forms. T congolense forms in the skin reactions had a well developed secretory reticulum, small mitochondria and lacked large lipid inclusions compared to metacyclic and blood stream forms. The intracutaneous forms of T brucei had smaller mitochondria, the glycosomes were of more uniform size and the rough endoplasmic reticulum was less developed than in metacyclic or blood stream forms.

SUMMARYThe feeding behaviour ofGlossina morsitans morsitansinfected withTrypanosoma vivax, T. congolenseorT. bruceiwas studied and compared with that of uninfected control tsetse. The following parameters were measured. (1) Mean number of feeds taken in 20 consecutive days; (2) mean rate of blood meal intake in μg/sec of the total days fed; (3) mean weight of blood meal intake/day of the total days fed; (4) mean total blood meal engorgement in 20 days; (5) mean number of probes/day of the days fed; (6) mean frequency of the multiple probe feeds and (7) mean number of pre-feeding probes of the multiple probe feeds. Evidence was found to suggest that the rosettes ofT. vivaxandT. congolensein the proboscis, and colonies ofT. bruceiparasites in the salivary glands, did not significantly alter the feeding behaviour of the vector.

Skin reactions similar to those induced by tsetse infected with Trypanosoma congolense were elicited in cattle at sites of intradermal inoculation of in vitro propagated parasites which morphologically resembled metacyclic trypanosomes. The time to detection of the reaction, the time to maximal size and the maximal size attained were dependent on the number of parasites inoculated, although it was possible to induce a skin reaction with as few as 20 trypanosomes. All cattle became infected with the initial detection of the skin reaction preceding parasitaemia by 3 to 7 days.

The metacyclic trypanosomes of a Trypanosoma brucei brucei clone (ILTat 2.1) were analysed with regard to their variable antigen (VAT) composition using monoclonal antibodies. The metacyclic population was antigenically heterogeneous. Despite the heterogeneity, however, the overall VAT composition of the metacyclic population appeared to be limited in number. A similar pattern of reactivity was observed when the monoclonal antibodies were tested on metacyclics of another clone (IL Tat 2.2) derived from a rabbit 30 days after infection with IL Tat 2.1 as well as those of the parent stock (STIB 247). The VAT characteristics of the metacyclics of this serodeme were consistent regardless of whether they were transmitted by Glossina morsitans morsitans or G.m. centralis. The monoclonal antibodies also reacted with some of the bloodstream VATs isolated within 72 h from cyclically infected mice. None of the monoclonal antibodies, however, reacted with metacyclics of a different stock (LUMP 227).

SUMMARYTo determine if, as is the case withTrypanosoma bruceiandT. congolense, serodemes ofT. vivaxcould be distinguished on the basis of immunity to the metacyclic stages of the parasite, attempts were made to immunize goats by infection with infected tsetse, followed by chemotherapy or eventual ‘self-cure’. Thirty goats were infected by tsetse with either clones or stocks ofT. vivaxfrom East or West Africa. Twenty-four goats were treated with diminazene aceturate (Berenil, Hoechst A.G.) 2–6 weeks after infection and 6 goats were allowed to self-cure. Infection, followed by treatment, induced immunity to a first homologous challenge by infected tsetse in only 2 of 24 goats (one immune to the East African stock, and the other to a clone of the West African stock). Immunity to a clone of the East African stock was induced in 3 or 4 animals after a second infection and treatment and in the fourth animal of the group following a third infection and treatment. One of 2 goats infected with the clone of the East African stock was immune to challenge at 16 weeks, following self-cure without treatment, and 1 of 4 goats infected with the parent stock was similarly immune when challenged at 40 weeks post-infection. Goats susceptible to infection with East AfricanT. vivaxshowed evidence of partial immunity by delayed pre-patent periods and depressed parasitaemias after challenge. Goats infected with the relatively more virulent West AfricanT. vivaxwere, however, completely susceptible to infection after homologous challenge, and showed only a slight delay in pre-patent period. A similar result was obtained in a further 8 goats primed and challenged by large numbers of tsetse (20 or 100 infected tsetse/goat) with the West AfricanT. vivax. In further experiments using a very short treatment interval, infections following challenge were clearly shown to be the result of a lack of immunity rather than relapse following treatment. Lytic antibody activity to cultured metacyclic trypanosomes could not be detected during infection but such activity against bloodstream forms was detected after 2 weeks of infection. It is suggested that the primary reason for the erratic induction of immunity toT. vivaxemploying this methodology is the low number of metacyclics transmitted by infected tsetse, and thus poor antigenic stimulus encountered by goats upon tsetse challenge.

To examine the influence of an established infection on subsequent challenge with another unrelated trypanosome serodeme, cattle were subjected to two challenges, 5 to 6 wk apart, with unrelated isolates of Trypanosoma congolense. The primary infection inhibited the establishment of the second infection despite the initial absence of detectable antibody to the trypanosomes used for the second challenge. This was true whether the second challenge consisted of bloodstream forms of the parasite or metacyclics from infected Glossina m. morsitans. Rechallenge with bloodstream forms resulted in a slight antibody response, which was only detectable by immunofluorescence and was much less than in the challenge controls. Although animals subjected to the second challenge by tsetse flies showed no appreciable increase in parasitemia and, in most instances, no chancre reaction at the site where the tsetse bit, they developed readily detectable neutralizing antibody to the metacyclic trypanosomes. That this interference effect was not the result of specific immunity and required an active infection was confirmed by the finding that when infected animals were treated with Berenil prior to rechallenge, they were fully susceptible to the infection.

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.

Ten Boran steers were infected with Trypanosoma vivax, transmitted by Glossina morsitans centralis; five steers with a T vivax clone from Nigeria and five with a T vivax clone from Kenya. Eleven days after infection all 10 animals were treated with 0.5 mg kg-1 isometamidium chloride. Four steers infected with the Nigerian T vivax and all five infected with the Kenyan T vivax were completely cured. When different steers received a single prophylactic dose of 0.5 mg kg-1 isometamidium chloride and subjected to monthly tsetse-transmitted challenge with the same T vivax clones, complete protection was afforded for at least two months against challenge with the Nigerian T vivax, but for less than one month against the Kenyan T vivax. The findings indicate that the level of sensitivity of a T vivax population to the prophylactic activity of isometamidium chloride cannot be concluded from sensitivity studies based on the therapeutic action of the drug.

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.

Specific antisera against sera of 46 species of vertebrates were prepared. The antisera to 21 Bovidae species were raised in goats except the antiserum to goat serum which was raised in sheep. The antisera to 3 Suidae species were produced either in domestic pigs or warthogs, while antisera to most of the other vertebrate species were raised in rabbits. The antisera were used in an enzyme-linked immunosorbent assay (ELISA) to identify the source of bloodmeals ingested by teneral and non-teneral tsetse at different time intervals after feeding. The bloodmeal donors were identifiable in 100% of the teneral tsetse up to 40 h post-feeding and in 87.5% in those tested up to 74 h post-feeding. Non-teneral tsetse digested the species-distinguishing bloodmeal components faster than the tenerals. Bloodmeals could be identified in 100% non-tenerals at 20 h post-feeding but only 67.5% and 50% of the bloodmeals could be identified 40 h and 74 h post-feeding, respectively. The antisera were also able to identify mixed bloodmeals from closely related species.

A comparison was made of the susceptibility of buffaloes, cattle and goats to infection with Trypanosoma vivax transmitted either by Glossina morsitans centralis or by syringe inoculation. Three different isolates of T vivax (two from East Africa, one from West Africa) were used to compare skin reactions, parasitaemia, anaemia and the development of trypanosome-specific antibodies in buffaloes, cattle and goats. African buffaloes reared in captivity in an area free from trypanosomiasis proved to be highly resistant to infection with the three stocks of T vivax tested, irrespective of whether infection was by tsetse transmitted metacyclic forms or by intradermal or intravenous inoculation of bloodstream forms of the parasite. The bites of 19 tsetse infected with a West African T vivax stock did not cause local skin reactions, detectable bloodstream infections or antibody responses in two buffaloes. Following the bites of 120 tsetse flies infected with the same stock, two different buffaloes showed no local skin reactions, but had detectable bloodstream infections without showing signs of anaemia. Cattle and goats infected in a similar way showed severe local inflammatory skin reactions, high levels of parasitaemia and severe anaemia. The two East African stocks of T vivax caused no local skin reactions and only a transient parasitaemia in buffaloes following tsetse-transmitted infection or intradermal inoculation of bloodstream forms. On the other hand, cattle and goats infected with the East African stocks showed high parasitaemias but local skin reactions only occurred in the goats.(ABSTRACT TRUNCATED AT 250 WORDS)