Search

To understand the circulation and the spread of Trypanosoma congolense genotypes in animals of Fontem in the southwest region of Cameroon, T. congolense forest and T. congolense savannah were investigated in 397 domestic animals in eight villages. Out of the 397 domestic animals, 86 (21.7%) were found infected by trypanosomes, using the capillary tube centrifugation test. The PCR with specific primers identified 163 (41.1%) and 81 (20.4%) animals infected by T. congolense forest and T. congolense savannah, respectively; showing for the first time the circulation of T. congolense savannah in the Fontem region. No infection with T. congolense savannah was found in pigs whereas goats and sheep were infected by T. congolense forest and/or T. congolense savannah. The prevalence of trypanosomes varied significantly amongst villages and animal species. The genotyping of T. congolense forest positive samples using microsatellites markers showed that multiple genotypes occurred in 27.2% (44/163) of animals sampled, whereas single genotypes were found in 73.8% (119/163) of samples. Some alleles were found in all animal species as well as in all villages and were responsible for major genotypes, whereas others (rare alleles) were identified only in some animals of few villages. These rare alleles were characteristic of specific genotypes, assimilated to minor genotypes which can be spread in the region through tsetse flies. The microsatellite markers show a low genetic variability and an absence of sub-structuration within T. congolense forest. The analysis of the microsatellite data revealed a predominant clonal reproduction within T. congolense forest. Pigs were the animal species with the highest number of different genotypes of T. congolense forest. They seem to play an important epidemiological role in the propagation and spread of different genotypes of T. congolense., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The tsetse fly (Diptera: Glossinidae), the vector of trypanosomes causing human and animal trypanosomiasis, harbors symbiotic microorganisms including the primary symbiont Wigglesworthia glossinidia, involved in the fly's nutrition and fertility, and the secondary symbiont Sodalis glossinidius, involved in the trypanosome establishment in the fly's midgut. Both symbionts are maternally transmitted to the intrauterine progeny through the fly's milk gland secretions. In this study, we investigated the population dynamics of these symbionts during fly development. Wigglesworthia and Sodalis densities were estimated using quantitative PCR performed on Glossina palpalis gambiensis at different developmental stages. The results showed that the density of the primary Wigglesworthia symbiont was higher than that of Sodalis for all host developmental stages. Sodalis densities remained constant in pupae, but increased significantly in adult flies. The opposite situation was observed for Wigglesworthia, whose density increased in pupae and remained constant during the female adult stage. Moreover, Wigglesworthia density increased significantly during the transition from the pupal to the teneral stage, while mating had a contradictory effect depending on the age of the fly. Finally, tsetse fly colonization by both symbionts appears as a continuous and adaptive process throughout the insect's development. Last, the study demonstrated both symbionts of G. p. gambiensis, the vector of the chronic form of human African trypanosomiasis, to be permanent inhabitants of the colony flies throughout their life span. This was expected for the primary symbiont, Wigglesworthia, but not necessarily for the secondary symbiont, S. glossinidius, whose permanent presence is not required for the fly's survival. This result is of importance as Sodalis could be involved in the tsetse fly vector competence and may constitute a target in the frame of sleeping sickness fighting strategies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Most African trypanosome species are sensitive to trypanolytic factors (TLFs) present in human serum. Trypanosome lysis was demonstrated to be associated with apolipoprotein L-I (APOL1). Trypanosoma brucei (T. b.) gambiense and Trypanosoma brucei rhodesiense, the two human infective trypanosome species, have both developed distinct resistance mechanisms to APOL1 mediated lysis. Whereas T. b. rhodesiense resistance is linked with the expression of the serum resistance associated (SRA) protein that interacts with APOL1 inside the parasite lysosome, inhibiting its lytic action; T. b. gambiense resistance is rather controlled by a reduced expression of the parasite HpHb receptor, limiting APOL1 absorption by trypanosomes. Based on this last observation we hypothesised that variation in the host APOL1 environment could significantly alter T. b. gambiense growth and thus resistance/susceptibility to sleeping sickness. To test this hypothesis, we have measured blood APOL1 relative expression in HAT patients, uninfected endemic controls and serologically positive subjects (SERO TL(+)) that are suspected to control infection to parasitological levels that are undetectable by the available test used in the field. All RNA samples were obtained from medical surveys led in the HAT mangrove foci of Coastal Guinea. Results indicate that APOL1 expression is a complex trait dependant on a variety of factors that need to be taken into account in the analysis. Nevertheless, multivariate analysis showed that APOL1 expression levels were significantly higher in both HAT and SERO TL(+) subject as compared to endemic controls (p=0.006). This result suggests that APOL1 expression is likely induced by T. b. gambiense, but is not related to resistance/susceptibility in its human host., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Human African trypanosomosis, caused by Trypanosoma brucei gambiense, is a chronic disease, although various clinical patterns have been observed, from asymptomatic to acute forms. Since 2001 in Angola, 80% of patients have been found to be in the meningoencephalitic stage of the disease. The existence of an acute form of the disease caused by virulent strains of trypanosomes was suspected. To test this hypothesis, four sensitive and polymorphic microsatellite markers were used to characterize the trypanosome DNA extracted from the blood and cerebrospinal fluid of 100 patients in the meningoencephalitic stage. Twenty-three patients were found with mixed T. b. gambiense genotypes in the blood and/or cerebrospinal fluid. The absence of association between the number of infecting genotypes, the presence of neurological signs and white blood cell counts in the cerebrospinal fluid, seems to indicate, at least in the context of the present study, the absence of virulent strains. However, out of five patients who died from encephalopathy syndrome during treatment with eflornithine, three harbored multiple infections., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Trypanosoma brucei subspecies undergo establishment and maturation in tsetse flies mid-gut and salivary glands, respectively. Successful establishment of trypanosomes in tsetse mid-gut as well as their migration to saliva gland depends on the ability of these parasites to adapt rapidly to new environmental conditions and to negotiate the physical barriers. To identify subspecies specific genes which are differentially regulated during the establishment of T. brucei subspecies in tsetse flies mid-gut, a comparative genomic analysis between different T. brucei subspecies was performed using microarrays containing about 23040 T. brucei shotgun fragments. The whole genome analyses of RNA expression profiles revealed about 274 significantly differentially expressed genes between T. brucei subspecies. About 7% of the differentially regulated clones did not match to any T. brucei predicted genes. Most of the differentially regulated transcripts are involved in transport across cell membrane and also in the purines metabolism. The genes selectively up regulated in T. brucei gambiense and T. brucei rhodesiense (human infective T. brucei) like snoRNA and HSP70 are expressed in response to stress. The high failure rate in the process of establishment and maturation of T. brucei gambiense during cyclical transmission in tsetse flies may result from the incapacity of this parasite to regulate its growth due to the expression of a variety of chaperones or heat shock proteins. Genes selectively up regulated in T. brucei brucei like NT8.1 nucleoside/nucleobase transporters and S-adenosylmethionine synthetase may favour the establishment of this subspecies in tsetse mid-gut. These genes appear as potential targets for investigations on the development of vaccine blocking the transmission of trypanosomes in tsetse flies.

Although clearly demonstrated in Trypanosoma brucei, genetic exchange remains controversial in other trypanosome species. Recently, Morrison and co-workers applied a population-genetics analysis, and established the existence of mating in Trypanosoma congolense. Starting from this original discovery, we focus here on the important question of how mating is induced during the trypanosome life cycle and discuss the use of statistics to evidence this type of non-obligatory biological process., (2009 Elsevier Ltd. All rights reserved.)

Epidemiological surveys were conducted in two historical human African trypanosomiasis foci in South Cameroon, Bipindi and Campo. In each focus, three sampling areas were defined. In Bipindi, only Glossina palpalis was identified, whereas four species were identified in Campo, G. palpalis being highly predominant (93%). For further analyses, 75 flies were randomly chosen among the flies trapped in each of the six villages. Large and statistically significant differences were recorded between both (1) the prevalence of Sodalis glossinidius (tsetse symbiont) and the prevalence of trypanosome infection of the major fly species G. p. palpalis and (2) the respective prevalence of symbiont and infection between the two foci. Despite these differences, the rate of infected flies harbouring the symbiont was very similar (75%) in both foci, suggesting that symbionts favour fly infection by trypanosomes. This hypothesis was statistically tested and assessed, showing that S. glossinidius is potentially an efficient target for controlling tsetse fly vectorial competence and consequently sleeping sickness., (Copyright 2009 Elsevier B.V. All rights reserved.)

Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of microorganisms. Seven microsatellite markers were used here to characterize Trypanosoma brucei gambiense isolates from Central Africa sub-region in order to improve knowledge on the population genetic structure of this subspecies. These markers confirmed the low genetic polymorphism within Central African T. b. gambiense isolates from the same focus and strong differentiation between different foci. The presence of many multilocus genotypes of T. b. gambiense and the excess of heterozygotes found in this study play in favour of a clonal reproduction of this parasite. But some data may be indicative of a unique recombination event in one subsample. The high F(ST) value indicates low migration rates between T. b. gambiense subpopulations (foci). Very negative F(IS) suggests fairly small clonal population sizes of this pathogen in the different human trypanosomosis foci of Central Africa., (Copyright 2009 Elsevier B.V. All rights reserved.)

This paper reports the first evidence of the presence of bacteria, other than the three previously described as symbionts, Wigglesworthia glossinidia, Wolbachia, and Sodalis glossinidius, in the midgut of Glossina palpalis palpalis, the tsetse fly, a vector of the chronic form of human African trypanosomiasis in sub-Saharan African countries. Based on the morphological, nutritional, physiological, and phylogenetic results, we identified Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the midgut of the tsetse fly from Angola. Enterobacter spp. was the most frequently isolated. The role of these bacteria in the gut, in terms of vector competence of the tsetse fly, is discussed, as is the possibility of using these bacteria to produce in situ trypanolytic molecules.

Substantial differences have been observed between the cyclical transmission of three Trypanosoma brucei gambiense field isolates in Glossina palpalis gambiensis (Ravel et al., 2006). Differences in the pleomorphism of these isolates in rodent used to provide the infective feed to Glossina, could explain such results, since stumpy forms are preadapted for differentiation to procyclic forms when taken up in a tsetse bloodmeal. To assess this possibility, mice were immunosuppressed and inoculated intraperitoneally with the three isolates (six mice for each trypanosome isolate); then parasitaemia and pleomorphism were determined daily for each mouse. The three T. b. gambiense isolates induced different infection patterns in mouse. The parasitaemia peak was rapidly reached for all the isolates and maintained until mice death for two isolates, while the third isolate rapidly showed a falling phase followed by a second parasitaemia plateau. The proportion of the stumpy forms varied from 15% to 70% over the duration of the experiment and according to the isolate. One isolate, which displayed the highest proportion of stumpy forms and reached the stumpy peak at the onset of the falling phase of parasitaemia, was used to study the relationship between the proportion of stumpy forms and transmissibility to tsetse fly. The results indicated that the transmissibility of trypanosomes was not correlated to the proportion of non-dividing stumpy forms.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.

Despite the impact of some trypanosome species on human and livestock health, the full diversity of trypanosomes in Africa is poorly understood. A recent study examined the prevalence of trypanosomes among a wide variety of wild vertebrates in Cameroon using species-specific PCR tests, but six trypanosome isolates remained unidentified. Here they have been re-examined using fluorescent fragment length barcoding (FFLB) and phylogenetic analysis of glycosomal glyceraldehyde phosphate dehydrogenase gGAPDH and 18S ribosomal RNA (rDNA) genes. Isolates from a monkey (Cercopithecus nictitans) and a palm civet (Nandinia binotata) belonged to the Trypanosoma cruzi clade, known previously only from New World and Australian terrestrial mammals, and bats from Africa, Europe and South America. Of the four other isolates, three from antelope were identified as Trypanosoma theileri, and one from a crocodile as T. grayi. This is the first report of trypanosomes of the T. cruzi clade in African terrestrial mammals and expands the clade's known global distribution in terrestrial mammals. Previously it has been hypothesized that African and New World trypanosomes diverged after continental separation, dating the divergence to around 100 million years ago. The new evidence instead suggests that intercontinental transfer occurred well after this, possibly via bats or rodents, allowing these trypanosomes to establish and evolve in African terrestrial mammals, and questioning the validity of calibrating trypanosome molecular trees using continental separation.

To determine the tsetse fly host preferences in two sleeping sickness foci of southern Cameroon, four entomological surveys (two in each focus) were carried out. For the whole study, 4929 tsetse flies were caught: 3933 (79.8%) Glossina palpalis palpalis, 626 (12.7%) Glossina pallicera pallicera, 276 (5.6%) Glossina nigrofusca and 94 (1.9%) Glossina caliginea. One hundred and thirty-eight blood meals were collected and the origin of 118 (85.5%) meals was successfully identified: 38.4% from man, 23.9% from pig, 20.3% from sitatunga (Tragelaphus spekeii), 2.2% from sheep and 0.7% from golden cat (Profilis aurata). The number of Glossina palpalis palpalis with man blood meals is more important in the Human African Trypanosomiasis (HAT) focus showing endemic evolution (Campo) than in the focus (Bipindi) presenting a flare up of the disease. The consideration of both results of the prevalence of Trypanosoma brucei gambiense in vertebrate hosts and those of the tsetse fly host preferences indicates a wild animal reservoir of Gambian sleeping sickness and three transmission cycles (human, domestic and wild animals' cycles) in southern Cameroon HAT foci.

The pathogenic agent of human African trypanosomiasis (HAT) is a trypanosome belonging to the species Trypanosoma brucei s.l. Molecular methods developed for typing T. brucei s.l. stocks are for the most part not polymorphic enough to study genetic diversity within T. brucei gambiense (T. b. gambiense) group 1, the main agent of HAT in West and Central Africa. Furthermore, these methods require high quantities of parasite material and consequently are hampered by a selection bias of the isolation and cultivation techniques. In this study, we evaluated the potential value of microsatellite markers (eight loci) in the genetic characterisation of T. brucei s.l. compared to the multi-locus enzyme electrophoresis reference technique. Stocks isolated in Ivory Coast and reference stocks were used for this purpose. Microsatellite markers were shown to be polymorphic enough to evidence the existence of genetic diversity within T. b. gambiense group 1 and to show the existence of mixed infections. Furthermore, they were able to amplify trypanosome DNA directly from field samples without the usual culturing stages. While the ability of microsatellite markers to detect mixed infections in such field samples is currently being discussed, they appear to be useful to study the parasite population's geographical structure and may provide new insight into their reproductive mode, a topic that is still under debate. Thus, use of microsatellite markers will contribute to the study of the influence of parasite genetics in the diversity of responses to HAT and may contribute to the improvement of HAT molecular diagnosis.

Glossina palpalis is the main vector of human African trypanosomosis (HAT, or sleeping sickness) that dramatically affects human health in sub-Saharan Africa. Because of the implications of genetic structuring of vector populations for the design and efficacy of control campaigns, G. palpalis palpalis in the most active focus of sleeping sickness in Côte d'Ivoire was studied to determine whether this taxon is genetically structured. High and statistically significant levels of within population heterozygote deficiencies were found at each of the five microsatellite loci in two temporally separated samples. Neither null alleles, short allele dominance, nor trap locations could fully explain these deviations from random mating, but a clustering within each of the two samples into different genetic sub-populations (Wahlund effect) was strongly suggested. These different genetic groups, which could display differences in infection rates and trypanosome identity, were composed of small numbers of individuals that were captured together, leading to the observed Wahlund effect. Implications of this population structure on tsetse control are discussed.

In order to study the existence of a wild animal reservoir for Trypanosoma brucei gambiense in South Cameroon, blood was collected from wild animals in three human African trypanosomiasis foci and from a nonendemic control area. The 1142 wild animals sampled belonged to 36 different species pertaining to eight orders (407 primates, 347 artiodactyls, 265 rodents, 54 pangolins, 53 carnivores, 11 saurians and crocodilians, and five hyraxes). QBC and KIVI tests detected trypanosomes on 1.7% (13/762) and 18.4% (43/234) of animals examined, respectively. Using specific primers, T. brucei non-gambiense group 1 DNA was detected on 56 animals (4.9%). This infection rate was 5.3% in the endemic zone and 3.8% in the control zone. Of the 832 animals of the endemic zone, PCR revealed T. b. gambiense group 1 DNA in 18 (2.2%). These hosts included two rodents, two artiodactyls, two carnivores and two primates. T. b. gambiense group 1 was absent from animals from the nonendemic zone. A decrease in the prevalence of T. b. gambiense group 1 was observed in wild animals from the Bipindi sleeping sickness focus after a medical survey and vector control in this area. The epidemiological implications of these findings remain to be determined with further investigations.

Genetic studies of Trypanosoma brucei have been mainly based on rodent inoculation (RI) for isolation of trypanosome strains. However, Trypanosoma brucei gambiense is difficult to grow in rodents. The development and use of the Kit for In Vitro Isolation (KIVI) of trypanosomes has led to a better isolation success. However, some authors report a genetic monomorphism in T. b. gambiense, and the extensive use of the KIVI was suspected as being responsible for this low genetic diversity. In the present work, trypanosome stocks were isolated from both humans and pigs in an active sleeping sickness focus in Côte d'Ivoire. Two methods were simultaneously used for this purpose: KIVI and rodent inoculation. None of the human stocks grew in rodents. Some of the stocks originating from pigs could be isolated with both methods. Each of these stocks (from the same pig) showed a different isoenzymatic pattern according to the isolation method used. All the human stocks identified belonged to the major zymodeme 3 of T. b. gambiense group 1, whereas the stocks isolated from pigs belonged to a new group of zymodemes even if they were genetically closely related. These observations may have significant implications when analysing the population structure of T. brucei, and also raise again the question of the importance of the animal reservoir in Human African Trypanosomiasis (HAT).

We studied the subcellular distribution of cytochrome P450 and related monooxygenase activities in six regions of human brains removed at autopsy. The content of total cytochrome P450 was found to be at least nine times higher in the mitochondrial fraction than in the microsomes in all the regions studied. However, cytochrome P450-dependent enzymatic activities which are representative of different isoforms metabolizing exogenous molecules exhibited a microsomal prevalence, a situation previously observed in rat brain. The other drug-metabolizing enzymes catalysing functionalization and conjugation reactions, presented the following characteristics in human brain: (i) a low activity of NADPH-cytochrome P450 reductase, which also catalyses the reduction of some xenobiotics; (ii) a high specific activity of the membrane-bound epoxide hydrolase; (iii) among the enzymes catalysing conjugation reactions, 1-naphthol-UDP-glucuronosyltransferase activity was barely or not detectable, whereas the mean glutathione-S-transferase activity was 15 times higher than the activity measured in rat brain. The presence of several drug-metabolizing enzyme activities in human brain microvessels, and particularly the high activity of epoxide hydrolase, suggests a participation of these enzymes in the metabolic blood-brain barrier.