Thioester-containing proteins in the tsetse fly (Glossina) and their response to trypanosome infection.

Thioester-containing proteins (TEPs) are conserved proteins with a role in innate immune immunity. In the current study, we characterized the TEP family in the genome of six tsetse fly species (Glossina spp.). Tsetse flies are the biological vectors of several African trypanosomes, which cause sleeping sickness in humans or nagana in livestock. The analysis of the tsetse TEP sequences revealed information about their structure, evolutionary relationships and expression profiles under both normal and trypanosome infection conditions. Phylogenetic analysis of the family showed that tsetse flies harbour a genomic expansion of specific TEPs that are not found in other dipterans. We found a general expression of all TEP genes in the alimentary tract, mouthparts and salivary glands. Glossina morsitans and Glossina palpalis TEP genes display a tissue-specific expression pattern with some that are markedly up-regulated when the fly is infected with the trypanosome parasite. A different TEP response was observed to infection with Trypanosoma brucei compared to Trypanosoma congolense, indicating that the tsetse TEP response is trypanosome-specific. These findings are suggestive for the involvement of the TEP family in tsetse innate immunity, with a possible role in the control of the trypanosome parasite.

Aberrant use and poor quality of trypanocides: a risk for drug resistance in south western Ethiopia.

BACKGROUND: Trypanocidal drugs have been used to control African animal trypanosomosis for several decades. In Ethiopia, these drugs are available from both authorized (legal) and unauthorized (illegal) sources but documentation on utilization practices and quality of circulating products is scanty. This study looked at the practices of trypanocidal drug utilization by farmers and the integrity of active ingredient in trypanocides sold in Gurage zone, south western Ethiopia. The surveys were based on a structured questionnaire and drug quality determination of commonly used brands originating from European and Asian companies and sold at both authorized and unauthorized markets. One hundred farmers were interviewed and 50 drug samples were collected in 2013 (Diminazene aceturate = 33 and Isometamidium chloride = 17; 25 from authorized and 25 from unauthorized sources). Samples were tested at the OIE-certified Veterinary Drug Control Laboratory (LACOMEV) in Dakar, Senegal, by using galenic standards and high performance liquid chromatography. RESULTS: Trypanosomosis was found to be a major threat according to all interviewed livestock keepers in the study area. Diminazene aceturate and isometamidium chloride were preferred by 79% and 21% of the respondents respectively, and 85% of them indicated that an animal receives more than six treatments per year. About 60% of these treatments were reported to be administered by untrained farmers. Trypanocidal drug sources included both unauthorized outlets (56%) and authorized government and private sources (44%). A wide availability and usage of substandard quality drugs was revealed. Twenty eight percent of trypanocidal drugs tested failed to comply with quality requirements. There was no significant difference in the frequency of non-compliance between diminazene-based and isometamidium chloride products (P = 0.87) irrespective of the marketing channel (official and unofficial). However, higher rates of non-compliant trypanocides were detected for drugs originating from Asia than from Europe (P = 0.029). CONCLUSION: The findings revealed the presence of risk factors for the development of drug resistance, i.e. wide distribution of poor quality drugs as well as substandard administration practices. Therefore, it is strongly recommended to enforce regulatory measures for quality control of veterinary drugs, to expand and strengthen veterinary services and to undertake trypanocidal drug efficacy studies of wider coverage.

The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

Maturation of a Trypanosoma brucei infection to the infectious metacyclic stage is enhanced in nutritionally stressed tsetse flies.

We report on the effect of tsetse fly starvation on the maturation of an established Trypanosoma brucei brucei midgut infection, i.e., the development of procyclic infection into the infectious metacyclic parasites in the tsetse fly salivary glands. Glossina morsitans morsitans flies were nutritionally stressed 10 d after the uptake of a T. b. brucei-infected bloodmeal by depriving these flies from feeding for seven consecutive days, whereas the control fly group (nonstarved group) continued to be fed three times a week. After this period, both fly groups were again fed three times per week on uninfected rabbit. Thirty days after the infected bloodmeal, all surviving flies were dissected and examined for the presence of an immature midgut and a mature salivary gland trypanosome infections. Results showed a significantly increased proportion of flies with salivary gland infection in the nutritionally stressed fly group suggesting an enhanced maturation of the trypanosome infection. These data suggest that environmental factors that cause nutritional stress in a tsetse population do not only make tsetse flies significantly more susceptible to establish a midgut infection as was shown previously but also boost the maturation of these midgut infections.

Nutritional stress affects the tsetse fly's immune gene expression.

Tsetse-transmitted trypanosomiasis poses a serious threat to human and animal health in sub-Saharan Africa. The majority of tsetse flies (Glossina spp.) in a natural population will not develop a mature infection of either Trypanosoma congolense or Trypanosoma brucei sp. because of refractoriness, a phenomenon that is affected by different factors, including the tsetse fly's immune defence. Starvation of tsetse flies significantly increases their susceptibility to the establishment of a trypanosome infection. This paper reports the effects of nutritional stress (starvation) on (a) uninduced baseline levels of gene expression of the antimicrobial peptides attacin, defensin and cecropin in the tsetse fly, and (b) levels of expression induced in response to bacterial (Escherichia coli) or trypanosomal challenge. In newly emerged, unfed tsetse flies, starvation significantly lowers baseline levels of antimicrobial peptide gene expression, especially for attacin and cecropin. In response to trypanosome challenge, only non-starved older flies showed a significant increase in antimicrobial peptide gene expression within 5 days of ingestion of a trypanosome-containing bloodmeal, especially with T. brucei bloodstream forms. These data suggest that a decreased expression of immune genes in newly hatched flies or a lack of immune responsiveness to trypanosomes in older flies, both occurring as a result of fly starvation, may be among the factors contributing to the increased susceptibility of nutritionally stressed tsetse flies to trypanosome infection.

The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins.

The tsetse fly (Glossina spp.) is an obligate blood-sucking insect that transmits different human-pathogenic and livestock threatening trypanosome species in Africa. To obtain more insight in the tsetse salivary function, some general aspects of the tsetse fly saliva and its composition were studied. Direct pH and protein content measurements revealed a moderately alkaline (pH approximately 8.0) salivary environment with approximately 4.3 microg soluble proteins per gland and a constant representation of the major saliva proteins throughout the blood-feeding cycle. Although major salivary genes are constitutively expressed, upregulation of salivary protein synthesis within 48 h after the blood meal ensures complete protein replenishment from day 3 onwards. Screening of a non-normalised Glossina morsitans morsitans lambdagt11 salivary gland expression library with serum from a saliva-immunized rabbit identified three full-length cDNAs encoding for novel salivary proteins with yet unknown functions: a 8.3 kDa glycine/glutamate-rich protein (G. morsitans morsitans salivary gland protein Gmmsgp1), a 12.0 kDa proline-rich protein (Gmmsgp2), and a 97.4 kDa protein composed of a metallophosphoesterase/5'nucleotidase region with a glutamate/aspartate/asparagines-rich region (Gmmsgp3).

Cattle breeding, trypanosomosis prevalence and drug resistance in Northern Togo.

African Animal Trypanosomosis (AAT) is a major disease of cattle in Togo and its control is essentially based on chemotherapy. However, because of excessive use of trypanocides during the past decades, chemo-resistance in the parasites has developed. In order to assess the current situation of AAT and resistance to trypanocidal drugs in Northern Togo, a study was conducted on cattle from December 2012 to August 2013 in the regions of Kara and Savanes. An initial cross-sectional survey was carried out in 40 villages using the Haematocrit Centrifugation Technique (HCT). Out of these, 5 villages with a trypanosome prevalence of >10% were selected for a block treatment study (BT) with diminazene diaceturate (DA: 3.5mg/kg for a 14-day follow-up) and isometamidium chloride (ISM: 0.5mg/kg for a 28-day follow-up). Positive blood samples collected during the parasitological surveys and an equivalent number of negatives were further analyzed by PCR-RFLP for trypanosome species confirmation and molecular diagnosis of resistance to DA in Trypanosoma congolense. The results from 1883 bovine blood samples confirmed a high overall trypanosome prevalence of 10.8% in Northern Togo. PCR-RFLP revealed that T. congolense is the dominant pathogenic trypanosome species (50.5%) followed by T. vivax (27.3%), and T. brucei (16.2%). The BT showed varying levels of treatment failures ranging from 0 to 30% and from 0 to 50% for DA and for ISM respectively, suggesting the existence of resistant trypanosome populations in the study area. Our results show that AAT still represents a major obstacle to the development of cattle husbandry in Northern Togo. In areas of high AAT risk, a community-based integrated strategy combining vector control, rational use of trypanocidal drugs and improving the general condition of the animals is recommended to decision makers.

Effect of isometamidium chloride treatment on susceptibility of tsetse flies (Diptera: Glossinidae) to trypanosome infections.

Experiments were conducted to determine the effect of a single isometamidium chloride treatment of teneral tsetse flies, Glossina morsitans morsitans Westwood (Diptera: Glossinidae), on the subsequent susceptibility to an infection with Trypanosoma congolense or Trypanosoma brucei brucei. Flies were offered a first bloodmeal on sterile gamma-irradiated defibrinated bovine blood that contained either 10 or 100 microg ofisometamidium chloride/ml. Treated flies were subsequently infected with T. congolense IL 1180 or T. b. brucei AnTAR1 on day 3, 5, 10, or 20 posttreatment. To determine the effect of a single treatment with isometamidium chloride at 10 microg/ml on the fly's susceptibility to infection with isometamidium chloride-resistant trypanosome strains, treated flies were infected with one of two resistant isogenic T. congolense IL 1180 strains 3 d after the first feed. Results showed that a single isometamidium chloride treatment at 10 microg/ml blood sufficed to reduce significantly the fly's subsequent susceptibility to infection. Only 6.8% of the flies that were treated with isometamidium chloride developed a mature infection with T. congolense in the mouthparts compared with 34.3% of the control group. None of the flies that were administered isometamidium chloride and subsequently infected on day 3 or 6 with T. b. brucei developed a metacyclic infection in the salivary glands compared with 22.7% of the control flies. Likewise for the resistant T. congolense strains, a single treatment with isometamidium chloride significantly reduced the subsequent susceptibility to infection (6.5 and 33.5% of flies with metacyclic infections for treated and untreated flies, respectively). In practice and with respect to the release of sterile male flies to eradicate an isolated tsetse fly population, our results show that administering isometamidium chloride during the first bloodmeal (and before release) would significantly reduce the ability of these released males to transmit trypanosomes.

Drug quality analysis through high performance liquid chromatography of isometamidium chloride hydrochloride and diminazene diaceturate purchased from official and unofficial sources in Northern Togo.

Trypanocidal drugs remain the most accessible and thus commonly used means of controlling tsetse transmitted animal African trypanosomosis. In Togo, trypanocides are sold on official as well as unofficial markets, but the quality of these trypanocides is undocumented so a drug quality assessment study was conducted from May 2013 to June 2014. Trypanocides supplied by European, Indian and Chinese pharmaceutical companies and sold on official and unofficial markets in Togo were purchased. In total fifty-two trypanocides were obtained, 24 of these samples from official markets and 28 from unofficial markets made up of a total of 36 diminazene diaceturate and 16 isometamidium chloride hydrochloride samples. The samples were analysed in the reference laboratory of the OIE (World Organisation for Animal Health), Laboratory for the Control of Veterinary Medicines (LACOMEV) in Dakar which uses galenic testing and high performance liquid chromatography (HPLC) testing as standard reference analysis methods. The results revealed a high proportion of trypanocides of sub-standard quality on the Togolese market: 40% were non-compliant to these quality reference standards. All of the HPLC non-compliant samples contained lower amounts of active ingredient compared to the concentration specified on the packaging. Non-compliance was higher in samples from the unofficial (53.57%) than from the official markets (25%; p=0.04).The main drug manufacturers, mostly of French origin in the study area, supply quality drugs through the official legal distribution circuit. Products of other origins mostly found on illegal markets present a significantly lower quality.

Ability of trypanosome-infected tsetse flies (Diptera: Glossinidae) to acquire an infection with a second trypanosome species.

The epidemiology of human and animal trypanosomiasis is determined to a large extent by the number of infected tsetse flies in a specific area. In the field, a substantial proportion of infected flies carry mixed trypanosome infections. The way in which these tsetse flies acquire a mixed infection is not fully understood. In particular, the susceptibility of tsetse flies to sequential infection with trypanosomes is not well understood. Accordingly, laboratory studies were made of the effects of age and prior infection on the probability of Glossina morsitans morsitans (Westwood) developing an infection of Trypanosoma congolense and Trypanosoma brucei brucei after feeding on infected mice. Results of these experiments clearly showed that 20-30-d-old G. m. morsitans can still pick up and develop a mature infection in the mouthparts/hypopharynx for T. congolense or in the salivary glands for T. b. brucei. However, their ability to acquire infection was significantly lower compared with teneral flies. Furthermore, 20-30-d-old flies that already carry a mature T. congolense or T. b. brucei infection remained at least as susceptible to a secondary trypanosome infection compared with noninfected flies of the same age. The immunological and epidemiological repercussions of those findings are discussed.

PCR and microsatellite analysis of diminazene aceturate resistance of bovine trypanosomes correlated to knowledge, attitude and practice of livestock keepers in South-Western Ethiopia.

African Animal Trypanosomosis is threatening the agricultural production and cattle breeding more severely than any other livestock disease in the continent, even more since the advent of drug resistance. A longitudinal study was conducted from November 2012 to May 2013 in the Ghibe valley to evaluate diminazene aceturate (DA) resistance and assess livestock owner's perception of trypanocidal drug use. Four Peasant Associations (PAs) were purposively selected and the cattle randomly sampled in each PAs. At the beginning of the study (t0), 106 bovines positive for trypanosomes by the haematocrit centrifugation technique (HCT) and 119 negative control animals were recruited for six months follow-up using HCT, 18S-PCR-RFLP, DpnII-PCR-RFLP and microsatellite analysis. Prevalence of trypanosomosis was 18.1% based on the HCT technique and the mean PCV value was 23.6±5.1% for the 587 sampled cattle. Out of the 106 HCT positive, 64 (60.4%) were positive for the presence of trypanosomes using the 18S-PCR-RFLP. Species detection showed 38 (59.4%) Trypanosoma congolense savannah, 18 (28.1%) Trypanosoma vivax, 5 (7.8%) Trypanosoma theileri and 3 (4.7%) T. congolense Kilifi. Among the T. congolense savannah samples, 31 (81.6%) showed a DA resistant RFLP profile, 2 (5.3%) a mixed profile and 5 did not amplify using the DpnII-PCR-RFLP. A positive HCT had a significant effect on PCV (p<0.001) with the mean PCV value equal to 24.4±0.2% in the absence of trypanosomes and to 20.9±0.3% in the presence of trypanosomes. PCV increased significantly (p<0.001) with 4.4±0.5% one month after treatment. All T. congolense savannah type were analyzed using microsatellite markers TCM1, TCM3 and TCM4. The main events were new infections (40.0%) and relapses (37.5%) with cures lagging at 22.5%. In 10 purposively selected PAs a semi-structured questionnaire was used. The average herd size was the highest in Abelti PA (6.7±1.8 TLU) and the mean herd size was statistically different (p=0.01) in the 10 PAs. Trypanosomosis was designated as the main disease affecting cattle by 97% of the respondents. DA was used by 95.5% of the farmers though more than half of them (51.9%) were not familiar with isometamidium (ISM). There was a trend to overdose young small animals and to underdose large ones. Oxen were treated very frequently (nearly 20 times/year) and calves almost never. To improve the situation in the Ghibe valley, extension messages should be delivered to promote a rational drug use, improved livestock management and the application of strategic vector control methods.

Trypanosome-binding proteins of the tsetse flies Glossina palpalis gambiensis and G. morsitans morsitans.

In this paper we describe a new, selective approach to identify protein ligand-receptor interactions between an arthropod vector and the parasite it transmits. Biotinylated vector proteins were incubated with living parasites in physiological conditions. After extensive washing, the parasites were subjected to SDS-PAGE electrophoresis and the polypeptides were electroblotted onto nitrocellulose membrane. Staining with avidin-horseradish peroxidase revealed only biotin-labeled proteins from the vector which were bound to the parasite. A multitude of tissue-specific proteins of Glossina palpalis gambiensis and G. morsitans morsitans proteins, able to bind to cultured procyclic trypanosomes of Trypanosoma brucei spp., has been demonstrated. The relevance of these interactions in relation to the developmental journey of the trypanosome in the tsetse fly is briefly discussed.

Detection of African animal trypanosomes: the haematocrit centrifugation technique compared to PCR with samples stored on filter paper or in DNA protecting buffer.

The present study aimed at comparing the trypanosome specific 18S-PCR-RFLP using samples stored either on Whatman filter papers (PCR-RFLP-fp) or in a commercial cell lysis and DNA protecting buffer (PCR-RFLP-pb) with the haematocrit centrifugation technique (HCT), a method widely used for the diagnosis of African Animal Trypanosomosis. Out of 411 head of cattle, 49 (11.92%) (CI=8.95-15.45) scored positive for the presence of trypanosomes by HCT whereas 75 (18.25%) (CI=14.63-22.33) and 124 (30.17%) (CI=25.77-34.86) scored positive using PCR-RFLP-fp and PCR-RFLP-pb, respectively. Out of the 49 positives by HCT, 14 (28.57%) (CI=16.58-43.26) and 28 (57.14%) (CI=42.21-71.18) were concordant by PCR-RFLP-fp and PCR-RFLP-pb, respectively. None of the PCR techniques detected parasites from the Trypanozoon group. Although HCT detected more cases of Trypanosoma vivax (33), species identification using PCR-RFLP-fp and PCR-RFLP-pb were significantly different (p<0.001) from the HCT technique. The use of DNA protective buffer is thus recommended as the output of the PCR-RFLP-pb is improved and the risk of contamination between samples is reduced.

Baseline survey of animal trypanosomosis in the region of the Boucle du Mouhoun, Burkina Faso.

In view of gathering baseline information about the prevalence of animal trypanosomosis, the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) funded a cross sectional survey in the region of the Boucle du Mouhoun which constitutes the Northern limit of the tsetse distribution in Burkina. This cross sectional study was carried out in 53 villages located in the six provinces of the region. A total of 2002 cattle, 1466 small ruminants and 481 donkeys were sampled. This survey showed that about 25% of the cattle had been treated with trypanocidal drugs within 3 months before the survey compared to 3% and 0.42% for the small ruminants and donkeys, respectively. Parasitological prevalence in cattle was low: 0.77% (95% C.I. 0.30-1.95%). No goats and three donkeys were found infected with trypanosomes. Infections were mainly due to Trypanosoma vivax (75.0%) with cases of Trypanosoma congolense (25.0%). In cattle, the serological prevalence of trypanosomosis, for the entire region of the Boucle du Mouhoun, was 34.2% (95% C.I. 26.1-43.4%). For sheep, goats and donkeys, the prevalence were of 20.9% (95% C.I. 12.2-33.5%), 8.5% (95% C.I. 5.7-12.5%) and 5.8% (95% C.I. 3.9-8.6%), respectively. The age and distance to the river were the two main risk factors associated with seropositivity.

Ghibe river basin in Ethiopia: present situation of trypanocidal drug resistance in Trypanosoma congolense using tests in mice and PCR-RFLP.

A cross-sectional study was carried out in the Ghibe valley from August to October 2010. 411 head of cattle were sampled in eight villages for buffy coat examination (BCE) and blood spots were collected from each animal for trypanosomose diagnosis by 18S-PCR-RFLP and diminazene aceturate (DA) resistance by Ade2-PCR-RFLP. Three villages were selected in a zone where trypanosomosis control operations are currently on-going whereas the other 5 villages were located outside these control operations. Twenty-four samples (5.84%) were diagnosed positive for Trypanosoma congolense by BCE and injected in mice for further characterization. Twelve of those isolates successfully multiplied in mice and were tested by an in vivo mouse test for diminazene (DA) (10 and 20mg/kg B.W.) and isometamidium (ISM) (1mg/kg B.W.) resistance. All were shown to be resistant to both drugs at all doses. The use of the Ade2-PCR-RFLP on these isolates confirmed their DA-resistance profile. Seventy-three of the collected blood spots (17.8%) were diagnosed positive for T. congolense by 18S-PCR-RFLP of which 37 (50.7%) gave amplification products with the Ade2-PCR-RFLP. Here, 35 (94.6%) showed a resistant profile, 1 (2.7%) a sensitive profile and 1 (2.7%) a mixed profile. The data were analysed by logistic regression model and the relapsing time in mice tests was assessed using the Cox regression model. There was no significant intervention effect (P=0.83) with odds ratio equal to 1.21 when using the BCE data. 18S-PCR-RFLP test also showed no significant intervention effect (P=0.60) with odds ratio equal to 1.43. The hazard ratio of getting parasitaemic after treatment with DA at 20mg/kg B.W. compared to the control group was 0.38 which differs significantly from one (P<0.001). Relapsing time after treatment with DA 10mg/kg B.W. or ISM 1mg/kg B.W. was also significantly longer than the prepatent period of the control group. The situation of drug resistance in the Ghibe valley is further discussed.

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

Field detection of resistance to isometamidium chloride and diminazene aceturate in Trypanosoma vivax from the region of the Boucle du Mouhoun in Burkina Faso.

A longitudinal study assessed the chemoresistance to isometamidium chloride (ISM) and diminazene aceturate (DA) in the region of the Boucle du Mouhoun in Burkina Faso. A preliminary cross-sectional survey allowed the identification of the 10 villages with the highest parasitological prevalences (from 2.1% to 16.1%). In each of these 10 villages, two herds of approximately 50 bovines were selected, one being treated with ISM (1mg/kg b.w.) and the other remaining untreated as control group. All animals (treated and untreated herds) becoming infected were treated with DA (3.5mg/kg b.w.). In total, 978 head of cattle were followed up. Fortnightly controls of the parasitaemia and PCV were carried out during 8 weeks. The main trypanosome species was Trypanosoma vivax (83.6%) followed by Trypanosoma congolense (16.4%). In two villages, less than 25% of the control untreated cattle became positive indicating no need to use prophylactic treatment. These two villages were not further studied. Resistance to ISM was observed in 5 of the remaining 8 villages (Débé, Bendougou, Kangotenga, Mou and Laro) where the relative risk (control/treated hazard ratios) of becoming infected was lower than 2 i.e. between 0.89 (95% CI: 0.43-2.74) and 1.75 (95% CI: 0.57-5.37). In contrast, this study did not show evidence of resistance to DA in the surveyed villages with only 8.6% (n=93) of the cattle relapsing after treatment. Our results suggest that because of the low prevalence of multiple resistances in the area a meticulous use of the sanative pair system would constitute the best option to delay as much as possible the spread of chemoresistance till complete eradication of the disease by vector control operations.

Nutritional stress of adult female tsetse flies (Diptera: Glossinidae) affects the susceptibility of their offspring to trypanosomal infections.

The epidemiology of tsetse-transmitted trypanosomiasis depends, among other factors, on the proportion of infected flies in a tsetse population. A wide range of intrinsic and extrinsic factors seem to determine the ability of a tsetse fly to become infected and to transmit the parasite. In this paper, we investigated the effect of nutritional stress of reproducing female Glossina morsitans morsitans on the susceptibility of their offspring to trypanosomal infections. Adult female flies that were nutritionally stressed by feeding only once a week, produced pupae with a significant lower weight and offspring with a significant lower fat content as well as a lower baseline immune peptide gene expression. Moreover, infection experiments showed that the emerging teneral flies were significantly more susceptible to a Trypanosoma congolense or Trypanosoma brucei brucei infection than flies emerging from non-starved adult females. These findings suggest that in the field, substantial nutritional stress of adult tsetse flies, as is often experienced during the hot dry season, can increase significantly the vectorial capacity of the emerging teneral flies and thus result in an increased infection rate of the tsetse population.

The effect of starvation on the susceptibility of teneral and non-teneral tsetse flies to trypanosome infection.

Transmission of vector-borne diseases depends largely on the ability of the insect vector to become infected with the parasite. In tsetse flies, newly emerged or teneral flies are considered the most likely to develop a mature, infective trypanosome infection. This was confirmed during experimental infections where laboratory-reared Glossina morsitans morsitans Westwood (Diptera: Glossinidae) were infected with Trypanosoma congolense or T. brucei brucei. The ability of mature adult tsetse flies to become infected with trypanosomes was significantly lower than that of newly emerged flies for both parasites. However, the nutritional status of the tsetse at the time of the infective bloodmeal affected its ability to acquire either a T. congolense or T. b. brucei infection. Indeed, an extreme period of starvation (3-4 days for teneral flies, 7 days for adult flies) lowers the developmental barrier for a trypanosome infection, especially at the midgut level of the tsetse fly. Adult G. m. morsitans became at least as susceptible as newly emerged flies to infection with T. congolense. Moreover, the susceptibility of adult flies, starved for 7 days, to an infection with T. b. brucei was also significantly increased, but only at the level of maturation of an established midgut infection to a salivary gland infection. The outcome of these experimental infections clearly suggests that, under natural conditions, nutritional stress in adult tsetse flies could contribute substantially to the epidemiology of tsetse-transmitted trypanosomiasis.

Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and proteolytic stress.

Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress-activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol-specific phospholipase-C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector.