Trypanosoma congolense: host responses following tsetse transmitted infection of Kilifi isolates in goats

East African x Galla goats, when infected with Trypanosoma congolense isolates from the Kilifi area of Kenya by Glossina morsitans centralis, did not develop the characteristic chancre reaction at the bite sites, whereas bites of tsetse infected with the cloned T. congolense IL.1180 from Serengeti, Tanzania, resulted in chancres in the same goats. Histological changes could not be observed in skin biopsies collected 8 or 9 days after infection with Kilifi isolates. However, all goats became parasitemic about 10 days after challenge. It is concluded that the absence of chancre development is a characteristic feature of T. congolense parasites from Kilifi. The isoenzyme analysis of clones of two T. congolense Kilifi isolates and the T. congolense clone IL.1180 indicated that they belong to different zymodemes. Neutralizing antibodies to homologous metacyclic variable antigen types were detected in six out of seven (86%) of the sera from goats infected with a clone or stock of a T. congolense Kilifi isolate, 20 days after infection. Goats primed by tsetse transmitted infection with a stock or clone of T. congolense from Kilifi and treated with Berenil were, in three out of eight cases (37%), not immune to homologous challenge. It is suggested that the reduced immune response to metacyclic variable antigen types could be a result of the absence of cellular infiltration, i.e., chancre development in the skin at the tsetse bite site. It is concluded that the use of the chancre reaction as a marker for serodeme analysis of recently isolated stocks of T. congolense from Kilifi was not feasible.     

Trypanosoma brucei: infectivity and immunogenicity of cultured parasites

Trypanosoma brucei brucei, derived from the salivary glands of infected tsetse flies (Glossina morsitans morsitans) and maintained in culture for over 4 years, were infective to both albino rats and tsetse flies. Virulence was markedly enhanced during the first passage in albino rats or tsetse flies. Irradiated cultured trypanosomes induced immunity to homologous challenge but not to tsetse fly or blood-induced challenge with the same stock.     

Feeding behaviour of Glossina pallidipes and g. morsitans centralis on Boran cattle infected with trypanosoma congolense or T. vivax under laboratory conditions

In field studies, tsetse flies (Diptera: Glossinidae) feed more successfully on cattle infected with Trypanosoma congolense Broden (Kinetoplastida: Trypanosomatidae) than on cattle infected with T. vivax Ziemann or uninfected cattle. Here we describe the first laboratory investigation of this phenomenon. In the first experiment, caged Glossina pallidipes Austen were fed for 1 and 5 min on a Boran steer infected with T. congolense clone IL 1180 and on an uninfected steer. Feeding success was recorded in this way five times over several weeks. The same protocol was subsequently used in three additional experiments with the following combinations: G. pallidipes and a steer infected with T. vivax stock IL 3913, G. morsitans centralis Machado and a steer infected with T. congolense, and G. morsitans centralis and a steer infected with T. vivax. The four experiments were replicated once, making eight experiments in total. In three experiments there was increased tsetse feeding success, measured at 1 min, after a steer became infected (T. congolense, two experiments and T. vivax, one experiment). Analysis of all data combined found no significant differences in tsetse feeding success on the different groups of cattle prior to infection, but after infection tsetse feeding success was significantly greater on the infected cattle (P< 0.001). Trypanosoma congolense infection led to a greater increase in tsetse feeding success than T. vivax infection. The increase in feeding success was not related to changes in the level of anaemia, skin surface temperature or parasitaemia. A possible explanation is the effects of trypanosome infection on cutaneous vasodilation and/or blood clotting in infected cattle. When allowed to feed for 5 min, nearly all tsetse engorged successfully and effects of cattle infection on feeding success were not found.     

Studies on the transmission of a West African stock of Trypanosoma vivax to rabbits,rats, mice and goats by Glossina morsitans morsitans and G. m. centralis

The cyclical transmission of a West African stock of Trypanosoma vivax by Glossina morsitans morsitans and G. m. centralis was studied. It was possible to transmit this parasite amongst rabbits, rats, mice and goats. Whereas goats and mice succumbed to the disease very rapidly, rats and rabbits showed scant and transient parasitaemia and frequently suppressed the infection. A trypanosome challenge using single infected tsetse showed that goats became infected much more readily than mice. The infected vector can transmit the infection throughout its life but not at every feed.     

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.     

Antigenic analysis by immunofluorescence of in vitro-produced metacyclics of Trypanosoma brucei and their infections in mice

The antigenic types in populations of Metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms with heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

Comparative study on the susceptibility of different laboratory strains of Glossina species to Trypanosoma simiae

Abstract. 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.     

Infectivity of monomorphic and pleomorphic trypanosoma brucei stocks cultivated at 28 C with various tsetse fly tissues

Noninfective procyclic forms of Trypanosoma brucei stocks derived from the pleomorphic EVE 10 were cultivated at 28 C in Cunningham's liquid medium in the presence of head-salivary gland, alimentary tract, and abdominal body wall explants of Glossina morsitans morsitans. After 8 to 10 days of cultivation some of the procyclic forms transformed into metacyclic stages infective for mice. Infectivity persisted for varying periods up to 66 days, when the experiments were terminated. Only 10 explants of alimentary tract or abdominal body wall tissues were required in the flasks to render the culture infective for most of the mice inoculated. Similar trypanosome suspensions grown with 10 head-salivary gland explants produced an infection on only one occasion. Cultures of procyclic organisms derived from the monomorphic stock 427 grown inthe presence of all three types of tsetse fly explants produced only sporadic infections in mice. Metacyclic forms failed to develop in trypanosome populations of stock EVE 10 cultivated at 28 C in the liquid medium alone or supplemented with mouse embryo tissues.     

Evaluation of insecticide-treated cattle as a barrier to re-invasion of tsetse to cleared areas in northeastern Zimbabwe

A field trial in Zimbabwe investigated the efficacy of insecticide-treated cattle as a barrier to prevent the re-invasion of tsetse, Glossina morsitans and G. pallidipes (Diptera: Glossinidae), into cleared areas. The original tsetse barrier consisted of insecticide-treated odour-baited targets, at an operational density of four to five targets per km2, supported by insecticide-treatments of cattle with either deltamethrin dip (Decatix, Coopers) at two-weekly intervals, or deltamethrin pouron (Spoton, Coopers) at monthly intervals, in a band approximately 20 km wide from the re-invasion front. Tsetse catch, and trypanosomiasis incidence in nine sentinel herds was recorded for 7-8 months, respectively, before the targets were removed, leaving only the insecticide treatment of the local cattle to stem the re-invasion of tsetse. After the removal of the target barrier, the tsetse readily invaded the trial area and the incidence of trypanosomiasis in sentinel herds increased, while their PCVs decreased. After seven months without the targets in place, trypanosomiasis prevalence in the local stock had reached alarmingly high levels; the trial was terminated prematurely and the target barrier re-deployed. Immediately after the re-deployment of the target barrier, the tsetse catch in the trial area reverted to acceptable levels along the re-invasion front, and trypanosomiasis incidence in the sentinel cattle decreased. It is concluded that, under the conditions of the field trial, the insecticidal treatment of local cattle did not in itself form an effective barrier to tsetse re-invasion. By contrast, the target barrier performed as was predicted by mathematical and experimental analysis, and readily cleared the tsetse infestation and reduced trypanosomosis incidence in the trial area.     

Unexpectedly slow homogenisation within a repetitive DNA family shared between two subspecies of tsetse fly

Summary Repetitive DNA families in sexual species are subject to a variety of turnover mechanisms capable of homogenising newly arising mutations. Very high levels of homogeneity in DNA families in some species ofDrosophila indicate that the rate of turnover is fast relative to that of mutation. To gauge the generality of such phenomena, we cloned and sequenced individual members of homologous repetitive DNA families from two subspecies of tsetse fly,Glossina morsitans centralis andG. morsitans morsitans. Unexpectedly high levels of variation were found within each subspecies, averaging 24% and 31%, respectively. Contiguous repeats and repeats cloned at random were comparably divergent. Nevertheless, it was possible to identify three instances of apparent homogenisation, each being, remarkably, of an insertion/deletion nature. We conclude that the rate of turnover in the tsetse families is comparable to that of most mutations, and discuss the possible parameters affecting flux in these families.     

Behaviour of tsetse flies (Glossina) in host odour plumes in the field

ABSTRACT. In Zimbabwe, studies were made of the responses of Glossina pallidipes Austen and G.morsitans morsitans Westwood to artificial host odour using an incomplete ring of electrocuting nets. In a plume of synthetic host odour tsetse flew generally upwind, with 50–60% flying within 35^o of due upwind. More than 80% of tsetse flew at < 50 cm above ground level. Upon losing contact with odour they executed a reverse turn within about 2 m, and upon regaining contact they turned upwind. There were no clear differences in the responses of G.m.morsitans and G.pallidipes. Using electrocuting nets lying horizontally on the ground it was found that tsetse landed in the vicinity of the odour source, the propensity to land being greater for G.pallidipes than for G.m, morsitans , greater for immature than mature flies, and greater for males than females.     

Seasonal variations in the distribution and abundance of the tsetse fly,Glossina morsitans morsitans in eastern Zambia

The seasonal changes in the distribution of Glossina morsitans morsitans Westwood (Diptera: Glossinidae) and its main host, cattle, were examined in a cultivated area of the plateau of eastern Zambia. During four consecutive years, the tsetse and cattle populations were monitored along a fly-round transect traversing the two main vegetation types in the study area. These were miombo, a one-storied open woodland with the genera Brachystegia and Julbernardia dominant, and munga, a one- or two-storied woodland where the principal tree genera were Acacia, Combretum and Terminalia. Concurrently, a capture/mark/release/recapture (CMRR) exercise was conducted along two other transects also traversing both vegetation types. The index of apparent abundance of tsetse (IAA) in miombo increased at the beginning of the rainy season (November), reached its peak at the end of the rainy season (April) and was low during the cold season (May to late August), but especially the hot dry season (September to late October). The IAA of tsetse in munga showed a pattern that was the reverse of that in miombo. The seasonal changes in the IAA of tsetse in both vegetation types were in accordance with changes in the movement patterns of tsetse between the two vegetation type as observed using CMRR. The distribution and abundance of cattle along the transect also showed a seasonal trend. This was especially so in munga, during the first three years of observations, where cattle abundance increased gradually from June onwards, reached a maximum at the end of the hot dry season (October-November) and declined steeply at the start of the rainy season (November-December). In both vegetation types, the monthly mean IAA of tsetse was positively correlated with the abundance of cattle in the previous month. It is concluded that the distribution of tsetse in cultivated area of the eastern plateau of Zambia undergoes substantial seasonal changes, which can partly be attributed to changes in the distribution of cattle. The implications of these observations for the control of tsetse are discussed.     

SDS-PAGE analysis of surface proteins and antigens evidences high heterogenity in natural clones of Trypanosoma cruzi, correlated with isoenzyme variability

Surface protein and surface antigen patterns of 19 Trypanosoma cruzi laboratory clones, representing 17 different isozymic profiles (zymodemes), were compared by SDS-PAGE analysis. Surface protein patterns were found to be complex and heterogeneous. According to the number of common bands, we calculated similarity coefficients of surface protein patterns on the one hand, and of surface antigen patterns on the other hand, for 33 stock pairwise comparisons. In both cases, these coefficients were statistically correlated to the isozyme index of genetic identity. Such a correlation between independent genetic markers favours the clonal structure of T. cruzi natural populations previously evidenced. Moreover, we did not observe any notable differences in the surface antigen pattern among 4 T. cruzi cloned stocks precipitated by homologous as well as heterologous hyperimmune sera. The immunological significance of the molecular weight variability in surface antigen patterns among different zymodemes is discussed.     

Genetic relationships between subspecies of the tsetse fly Glossina morsitans inferred from variation in mitochondrial DNA sequences

A 750 base pair segment of DNA from the tsetse fly Glossina morsitans morsitans was isolated by means of molecular cloning. It was shown by DNA hybridization to have substantial sequence homology with a defined region of the mitochondrial genomes of several Drosophila species. When used as a probe against DNA prepared from single tsetse flies, the cloned sequence revealed local restriction site variation between members of the G. morsitans subspecies complex. This feature was used to demonstrate maternal inheritance of the sequence in progeny of hybrid crosses and to assemble comparative restriction maps for a 3-kilobase segment of each mitochondrial genome. The data obtained from these exercises point to a higher genetic identity between G. m. morsitans and G. m. centralis than between either form and G. m. submorsitans.     

Use of deltamethrin 'pour-on' insecticide for the control of cattle trypanosomosis in the presence of high tsetse invasion

A deltamethrin 'pour-on' insecticide was applied monthly to over 2000 cattle exposed to a high challenge of drug-resistant trypanosomes and high tsetse re-invasion pressure in the Ghibe valley, south-west Ethiopia. Blood samples were taken monthly from an average of 760 cattle for determination of PCV and presence of trypanosomes. The area of the valley is approximately 350 km2 and the cattle grazed in roughly four locations covering about a quarter to half of the area. Two years before the trial commenced, Glossina morsitans submorsitans Newstead (Diptera: Glossinidae) began to invade the valley. Despite the use of the pour-on the mean apparent density of G. m. submorsitans continued to rise, and, during the 4 years of tsetse control, was more than three-fold higher than that recorded during the previous 18 months. Over the same period there was little change in the apparent density of Glossina pallidipes Austen (Diptera: Glossinidae). By contrast, the mean monthly prevalence of trypanosome infections in cattle over 36 months of age decreased from 38.3 to 29.0%, the incidence of new infections decreased from 26.6 to 16.0% (a reduction of 40%), and packed cell volume in cattle increased from 21.7 to 24.1%. Evidence of a change in apparent parasite transmission rate was demonstrated by regression of infection incidence in cattle on the logarithm of apparent density of G. m. submorsitans. Before the trial started the regression coefficient was 45.8 +/- 6.3 and this reduced to 9.2 +/- 2.5% incidence per log(e) (flies/trap/day) during the period of tsetse control. It was concluded that this indicated reductions in tsetse numbers in the immediate vicinities of cattle in a way that was not reflected in overall tsetse catches. Nevertheless, the comparatively high levels of trypanosome prevalence that persisted in the cattle demonstrates that, where invasion prevalence is high, treatment of small pockets of cattle will not eradicate tsetse. To achieve more significant reduction in trypanosome prevalence in cattle, integrated methods of control utilizing target barriers in the major routes of invasion will be needed.     

Identification of major soluble salivary gland proteins in teneral Glossina morsitans morsitans

Salivary glands of tsetse flies (Diptera: Glossinidiae) contain molecules that are involved in preventing blood clotting during feeding as well as molecules thought to be intimately associated with trypanosome development and maturation. Here we present a protein microchemical analysis of the major soluble proteins of the salivary glands of Glossina morsitans morsitans, an important vector of African trypanosomes. Differential solubilization of salivary proteins was followed by reverse-phase, high-performance liquid chromatography (HPLC) and analysis of fractions by 1-D gel electrophoresis to reveal four major proteins. Each protein was subjected to amino acid microanalysis and N-terminal microsequencing. A protein chemical approach using high-resolution 2-D gel electrophoresis and mass spectrometry was also used to identify the salivary proteins. Matrix-assisted, laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass mapping and sequencing, respectively. Sequence information and peptide mass maps queried against the NCBI non-redundant database confirmed the identity of the first protein as tsetse salivary gland growth factor-1 (TSGF-1). Two proteins with no known function were identified as tsetse salivary gland protein 1 (Tsal 1) and tsetse salivary gland protein 2 (Tsal 2). The fourth protein was identified as Tsetse antigen-5 (TAg-5), which is a member of a large family of anti-haemostatic proteins. The results show that these four proteins are the most abundant soluble gene products present in salivary glands of teneral G. m. morsitans. We discuss the possible functions of these major proteins in cyclical transmission of African trypanosomes.     

Obligate symbionts activate immune system development in the tsetse fly

Many insects rely on the presence of symbiotic bacteria for proper immune system function. However, the molecular mechanisms that underlie this phenomenon are poorly understood. Adult tsetse flies (Glossina spp.) house three symbiotic bacteria that are vertically transmitted from mother to offspring during this insect's unique viviparous mode of reproduction. Larval tsetse that undergo intrauterine development in the absence of their obligate mutualist, Wigglesworthia, exhibit a compromised immune system during adulthood. In this study, we characterize the immune phenotype of tsetse that develop in the absence of all of their endogenous symbiotic microbes. Aposymbiotic tsetse (Glossina morsitans morsitans [Gmm(Apo)]) present a severely compromised immune system that is characterized by the absence of phagocytic hemocytes and atypical expression of immunity-related genes. Correspondingly, these flies quickly succumb to infection with normally nonpathogenic Escherichia coli. The susceptible phenotype exhibited by Gmm(Apo) adults can be reversed when they receive hemocytes transplanted from wild-type donor flies prior to infection. Furthermore, the process of immune system development can be restored in intrauterine Gmm(Apo) larvae when their mothers are fed a diet supplemented with Wigglesworthia cell extracts. Our finding that molecular components of Wigglesworthia exhibit immunostimulatory activity within tsetse is representative of a novel evolutionary adaptation that steadfastly links an obligate symbiont with its host.     

Trypanosoma brucei 29-13 strain is inducible in but not permissive for the tsetse fly vector

Using green fluorescent protein as a reporter, we have shown that the strain 29-13 of Trypanosoma brucei, widely used for inducible down-regulation of mRNA, is inducible in, but not permissive for the tsetse flies Glossina palpalis gambiensis and Glossina morsitans morsitans. Within two weeks post-infection, 42% males and females of teneral and non-teneral tsetse flies harboured intestinal infections, yet not a single infection progressed into the salivary glands.     

Application of DNA markers to identify the individual-specific hosts of tsetse feeding on cattle

Primer sets for five different ungulate loci were used to obtain individual microsatellite DNA profiles for 29 Mashona cattle from a herd in Zimbabwe. There were 3-13 alleles for each locus and, using the entire suite of five loci, each animal within the herd, including closely related individuals, could be unequivocally distinguished. Wild-caught Glossina pallidipes Austen (Diptera: Glossinidae) were fed on specific cattle and the bloodmeal was profiled 0.5-72 h after feeding. The individual specific sources of the bloodmeals, including mixe meals produced by allowing tsetse to feed on two different cattle, were reliabl identified up to 24 h after feeding. The technique was used in field studies of hos selection by G. pallidipes and G. morsitans morsitans Westwood (Diptera Glossinidae) attracted to pairs of cattle. When the pair comprised an adult and a calf, 100% of meals were from the adult. For some pairs of adult cattle, tsetse were biased significantly towards feeding on one animal, whereas for other pairs there was no such bias. In general, feeding was greater on the animal known to have lower rate of host defensive behaviour. Results suggest that relatively slight differences in the inherent defensive behaviour of cattle produce large difference in host-specific feeding rates when the hosts are adjacent. For flies attracted to pair of cattle, < 2% contained blood from both hosts. The DNA profiling technique will be useful in studying the epidemiology of vector-borne diseases of livestock.     

Seasonal distribution and abundance of tsetse flies (Glossina spp.) in the Faro and Deo Division of the Adamaoua Plateau in Cameroon

Ten years after the large-scale tsetse control campaigns in the important cattle rearing areas of the Faro and Deo Division of the Adamaoua Plateau in Cameroon, the seasonal distribution and abundance of tsetse flies (Glossina spp.) were determined. During a period of 12 consecutive months (January-December 2005), the tsetse population was monitored along four trap transects consisting of a total of 32 traps and two flyround transects traversing the study area, which comprised the tsetse-infested valley, a buffer zone and the supposedly tsetse-free plateau. Throughout the study period, a total of 2195 Glossina morsitans submorsitans and 23 Glossina tachinoides were captured in the traps and 1007 G. m. submorsitans (78.8% male flies) were captured along the flyround transects. All G. tachinoides and almost all G. m. submorsitans were captured in the valley. Five G. m. submorsitans were captured in traps located in the buffer zone, whereas no flies were captured in traps located on the plateau. The index of apparent abundance (IAA) of G. m. submorsitans was substantially higher in the areas close to game reserves. In the remaining part of the valley, where wildlife is scarce and cattle are present during transhumance (dry season), the IAA of tsetse was substantially lower. In this part of the valley, the abundance of tsetse seemed to be associated with the presence of cattle, with the highest IAA during transhumance when cattle are present and the lowest apparent abundance during the rainy season when cattle have moved to the plateau. It is concluded that the distribution of tsetse in a large part of the valley undergoes substantial seasonal changes depending on the presence or absence of cattle. The repercussions of those findings for the control of tsetse in the valley and the probability of reinvasion of the plateau are discussed.