I. Maudlin

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

ABSTRACT. The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G.austeni Newstead and G.p.palpalis R‐D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A ‘refractory’ line of G.m.morsitans Westwood took 170 h to kill 50% infections while a ‘susceptible’ line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo.

In vitro cultivation of rickettsia-like-organisms from Glossina spp.

A method is described for the in vitro cultivation of the rickettsia-like-organisms (RLO) from Glossina spp. which are believed to be associated with susceptibility to trypanosome infection. Cultures of RLO were established by infecting a mosquito cell line (Aedes albopictus) with haemolymph taken from teneral flies. RLO from nine species of Glossina have been isolated and maintained in continuous culture using this technique.

The origins, dynamics and generation of Trypanosoma brucei rhodesiense epidemics in East Africa

The history of sleeping sickness in East Africa has provoked controversy not only about the origins and spread of the disease, but also the identity of the causative organisms involved. Molecular methodology(1) has shed new light on the genetic makeup of the organisms involved in recent epidemics. Here, Geoff Hide, Andrew Tait, Ian Maudlin and Susan Welburn discuss these new data in relation to previous theories about the origins of epidemics in East Africa which emphasized the importance of the introduction of new strains.

Rickettsia-like organisms and chitinase production in relation to transmission of trypanosomes by tsetse flies

Rickettsia-like organisms (RLO) from testse midguts and mosquito cell cultures showed high levels of endochitinase activity. A line of Glossina morsitans morsitans highly susceptible to midgut trypanosome infection and with high incidence of RLO infection showed significantly greater chitinolytic activity than G. austeni which had low RLO incidence and were correspondingly refractory to midgut infection. Midgut infection rates of Trypanosoma brucei rhodesiense in G. m. morsitans showed a dose-related increase when flies were fed N-acetyl-D-glucosamine (GlcNAc) in the infective meal and for 4 subsequent days. A model is proposed for susceptibility to trypanosome infection based on the generation of GlcNAc by RLO endochitinase activity in tsetse pupae inhibiting midgut lectin in teneral flies.

Transmission of African Trypanosomiasis: Interactions Among Tsetse Immune System, Symbionts, and Parasites

The threat posed to man and his domestic livestock by trypanosomiasis in Africa is related almost entirely to the distribution of infected tsetse flies. The natural infection rates of tsetse have long been used by those concerned with epidemiology and control to assess the risk or “challenge” created by the disease. Infection rates are determined by dissection and microscopy of testse; such examinations were (and still are) carried out across the fly belts of Africa—a typical example being the work of Pires et al. (102) who dissected more than 18,000 flies. The following infection rates were recorded for the three major pathogenic trypanosome groups: T. vivax 6%, T. congolense 2%, and no T. brucei infections (102). Jordan (60) brought together the results of field dissections such as these carried out in various parts of Africa between 1964 and 1972 and revealed that low levels of infection were the norm for all three major trypanosome groupings but especially for the brucei group, “mature” (salivary gland) infections of which were rarely seen. Localized “hot spots” were observed occasionally for vivax group infections.

The nature of the teneral state in Glossina and its role in the acquisition of trypanosome infection in tsetse.

Teneral Glossina morsitans morsitans from outbred and susceptible stocks infected with Trypanosoma (Nannomonas) congolense developed, respectively, three and six times higher midgut infection rates than flies of the same stock which had previously taken a bloodmeal. Non-teneral G. m. morsitans remained relatively refractory to infection when infected at subsequent feeds. Differences in susceptibility to midgut infection between teneral flies from susceptible and outbred lines of G. m. morsitans disappeared in non-teneral flies, showing that maternally inherited susceptibility to midgut infection is a phenomenon restricted to the teneral state of the fly. Laboratory reared G. m. morsitans were found to have become significantly more susceptible to trypanosome infection than wild flies from the population from which the colony was derived. The likely role of rickettsia-like organisms (RLO) in potentiating teneral susceptibility to midgut infection is discussed. The addition of the specific midgut lectin inhibitor D-glucosamine to the infective feed of non-teneral flies increased midgut infection rates to levels comparable with those achieved in teneral flies. It is concluded that the peritrophic membrane does not act as a barrier preventing non-teneral flies becoming infected. The relative refractoriness of non-teneral flies suggests that they do not play a significant part in the epidemiology of Trypanozoon or T. congolense infections.

Trypanosoma brucei rhodesiense: Characterisation of stocks from Zambia, Kenya, and Uganda using repetitive DNA probes

We have previously described a system for characterising the relationships between trypanosome stocks of the T.brucei group based on Southern blotting with repetitive DNA probes followed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Parasitol. 39, 213-226, 1990). In this study, we extend this analysis to examine the relationships between trypanosome stocks isolated from major sleeping sickness foci in Zambia, Kenya, and Uganda. We show that the trypanosome strains responsible for disease in Zambia are quite distinct from those sampled from the Kenya/Uganda foci. Furthermore, the human serum resistant stocks isolated from the Kenya/Uganda foci which were isolated from man (or from animals) were found to form a tight group in the cluster analysis, while stocks isolated from nonhuman sources in the same area or stocks from elsewhere were found in separate groups. Thus, the human infective trypanosome strains found in these foci may have common origins and have, perhaps, arisen by clonal selection from a common source.

The kinetics of maturation of trypanosome infections in tsetse

Estimates of the time delay between the infective bloodmeal and maturation (incubation or maturation time) for 4 trypanosome stocks (2 Trypanozoon and 2 Trypanosoma congolense) show that maturation time in tsetse is not a parasite species-specific constant. The mean incubation time of a Trypanosoma brucei rhodesiense stock (EATRO 2340 - 18 days) was not significantly different from one T. congolense stock (SIKUDA88 - 15.5 days) but was significantly greater than another (1/148 FLY9 - 12.5 days). There was no significant difference in incubation times between male and female Glossina morsitans morsitans for any of the stocks but in both of the Trypanozoon stocks the proportion of female flies producing mature infections was significantly less than in males. However, estimates of gene frequency, assuming a model in which maturation is controlled by an X-linked recessive allele, gave inconsistent results indicating that maturation cannot be controlled by a single sex-linked gene. Maturation was shown to be a tsetse sex-dependent phenomenon in Trypanozoon but not in T. congolense infections. Incubation time was quite variable even for a single trypanosome stock (e.g., standard deviation of 5 days for one Trypanozoon stock); we discuss how this variability can affect disease transmission, and the interpretation of age-prevalence data.

An estimate of the size of the metacyclic variable antigen repertoire of Trypanosoma brucei rhodesiense

A group of 27 variable antigen type (VAT)-specific monoclonal antibodies (McAbs) have been made against metacyclic forms of a cloned stock of Trypanosoma brucei rhodesiense. In combination, these labelled in immunofluorescence 99.3% of trypanosomes in salivary probes from tsetse flies. The 0.7% of unlabelled trypanosomes were believed to be uncoated forms. The ability of a mixture of antibodies to kill metacyclics in vitro by complement-mediated lysis, thus neutralizing their infectivity for mice, was tested. The antibody mixture consisted of 24 McAbs plus 3 VAT-specific rabbit antisera. In 12 replicate experiments this mixture of antibodies prevented infection of mice. Parallel controls showed that neutralization was probably antibody-mediated and VAT specific. However, we have not been able to repeat these results on a long-term basis; this may be due to a loss of neutralizing activity by one of the McAbs. The successful neutralization experiments indicate that the number of VATs in the metacyclic repertoire of one stock of T. b. rhodesiense is limited to at most 27.

Midgut lectin activity and sugar specificity in teneral and fed tsetse

Abstract. . Midgut infection rates of Trypanosoma congolense in Glossina palpalis palpalis and of Trypanosoma brucei rhodesiense in Glossina pallidipes are potentiated by the addition of D+ glucosamine to the infective feed, but not to the levels of super‐infection reported for G. m. morsitans. G. p. palpalis and G.pallidipes are shown to possess two trypanocidal molecules: a glucosyl lectin which can be inhibited by D+ glucosamine and a galactosyl molecule inhibited by D+ galactose. Addition of both D+ glucosamine and D+ galactose to the teneral infective feed promotes super‐infection of the midguts of G.p.palpalis. The glucosyl lectin is specific for rabbit erythrocytes and is present in guts of fed G.m.morsitans and G.p.palpalis, titres of lectin activity do not increase substantially after the second bloodmeal. The galactosyl specific molecule does not show any erythrocyte specificity, although haemolytic activity is observed only in G.p.palpalis and not in G.m.morsitans. The presence of two trypanocidal molecules in some species of tsetse may account for the innate refractoriness of these flies to trypanosome infection.