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BackgroundDespite the morphological characterization established in the 1950s and 1960s, the identity of extant taxa that make upGlossina fuscipes(s.l.) in the Congo remains questionable. Previous claims of overlap betweenG. fuscipes(believed to beG. f. quanzensis) andG. palpalis palpalisaround Brazzaville city further complicate the taxonomic status and population dynamics of the two taxa. This study aimed to determine the phylogenetic relationships betweenG. fuscipes(s.l.) andG. p. palpalisand to assess genetic variation amongG. fuscipes(s.l.) populations in Congo Brazzaville.MethodsWe collected 263G. fuscipes(s.l.) from northern and central regions, and 65G. p. palpalisfrom southern part of the country. The mitochondrial cytochromecoxidase subunit 1 (cox1) gene was amplified using taxa-specific primer pairs. Sequence data were analyzed in DnaSP and Arlequin to assess the genetic diversity, differentiation and demographic history ofG. fuscipes(s.l.) populations.ResultsThe general BLAST analysis yielded a similarity of 99% forG. fuscipes(s.l.) andG. p. palpalis. BLASTn analysis forG. fuscipes(s.l.) showed > 98% identity with GenBank sequences forG. fuscipes(s.l.), with BEMB population showing 100% similarity withG. f. fuscipes.Glossina fuscipes(s.l.) populations showed high haplotype diversity (H = 46, Hd = 0.884), moderate nucleotide diversity ( = 0.012) and moderate (FST= 0.072) to high (FST= 0.152) genetic differentiation. Most of the genetic variation (89.73%) was maintained within populations. The mismatch analysis and neutrality tests indicated recent tsetse population expansions.ConclusionsPhylogenetic analysis revealed minor differences betweenG. fuscipes(s.l.) andG. p. palpalis.Genetic diversity ofG. fuscipes(s.l.) was high in the populations sampled except one. Genetic differentiation ranged from moderate to high among subpopulations. There was a restricted gene flow betweenG. fuscipes(s.l.) populations in the north and central part of the country. Genetic signatures based oncox1 showed recent expansion and recovery ofG. fuscipes(s.l.) populations from previous bottlenecks. To fully understand the species distribution limits, we recommend further studies involving a wider sampling scheme including the swampy Mossaka focus forG. fuscipes(s.l.) and the entire range ofG. p. palpalisin South Congo.

African animal trypanosomiasis (AAT) is a major constraint to livestock productivity, particularly in cattle and in camels. This chapter covers some general aspects of the arthropod vectors of animal trypanosomiasis, the tsetse flies Glossina spp., and to a lesser extent the biting flies. This chapter covers the classification, morphology, basic biology, and the eco-distribution of tsetse flies. The role of tsetse flies in disease epidemiology has also been reviewed. The elementary biology of these vectors is quite well known and elucidated. However, with advances in molecular and other biological techniques, new insights related to tsetse biology have been obtained. This chapter will revisit these basics and include some updated information emanating from research done in the recent past. The final part of the chapter is devoted to a brief discussion on biting flies, the vectors of T. evansi, which causes camel trypanosomiasis.

This study assessed the virulence of Trypanosoma evansi, the causative agent of camel trypanosomiasis (surra), affecting mainly camels among other hosts in Africa, Asia and South America, with high mortality and morbidity. Using Swiss white mice, we assessed virulence of 17 T. evansi isolates collected from surra endemic countries. We determined parasitaemia, live body weight, packed cell volume (PCV) and survivorship in mice, for a period of 60 days’ post infection. Based on survivorship, the 17 isolates were classified into three virulence categories; low (31–60 days), moderate (11–30 days) and high (0–10 days). Differences in survivorship, PCV and bodyweights between categories were significant and correlated (P < 0.05). Of the 10 Kenyan isolates, four were of low, five moderate and one (Type B) of high virulence. These findings suggest differential virulence between T. evansi isolates. In conclusion, these results show that the virulence of T. evansi may be region specific, the phenotype of the circulating parasite should be considered in the management of surra. There is also need to collect more isolates from other surra endemic regions to confirm this observation.

African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies ( Glossina spp.). In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice ( n = 6). We further determined the vectorial capacity of Glossina pallidipes , for each of the trypanosome isolates. Results showed that the overall pre-patent (PP) periods were 8.4 ± 0.9 (range, 4–11) and 4.5 ± 0.2 (range, 4–6) for T. congolense and T. brucei isolates, respectively ( p < 0.01). Despite the longer mean PP, T. congolense –infected mice exhibited a significantly ( p < 0.05) shorter survival time than T. brucei –infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection–causing T. congolense EATRO 1829 and chronic infection–causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments.

Experiments on infections caused by trypanosomes are widely performed in Swiss white mice through various inoculation routes. To better understand the effect of route of trypanosome inoculation on disease outcomes in this model, we characterised the virulence of two isolates, Trypanosoma brucei KETRI 2710 and T. congolense KETRI 2765 in Swiss white mice. For each of the isolates, five routes of parasite inoculation, namely intraperitoneal (IP), subcutaneous (SC), intramuscular (IM) intradermal (ID) and intravenous (IV) were compared using groups (n = 6) of mice, with each mouse receiving 1x104 trypanosomes. We subsequently assessed impact of the routes on disease indices that included pre-patent period (PP), parasitaemia levels, Packed Cell Volume (PCV), bodyweight changes and survival time. Pre-patent period for IP inoculated mice was a mean ± SE of 3.8 ± 0.2 and 6.5 ± 0.0 for the T brucei and T. congolense isolates respectively; the PP for mice groups inoculated using other routes were not significantly different(p> 0.05) irrespective of route of inoculation and species of trypanosomes. With ID and IP routes, parasitaemia was significantly higher in T. brucei and significantly lower in T. congolense infected mice and the progression to peak parasitaemia routes showed no significant different between the routes of either species of trypanosome. The IM and ID routes in T. congolense inoculations, and IP and IV in T. b. brucei induced the fastest and slowest parasitaemia progressions respectively. There were significant differences in rates of reduction of PCV with time post infection in mice infected by the two species and which was more pronounced in sc and ip injected mice. No significant differences in mice body weight changes and survivorship was observed between the routes of inoculation. Inoculation route therefore appears to be a critical determinant of pathogenicity of Trypanosoma congolense and Trypanosoma brucei brucei in murine mouse model of African trypanosomiasis.

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10−6 (equivalent to 1 parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5–10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.

Chemical (anaesthesia) and manual techniques are commonly used to restrain mice during vector-mediated parasite transmission experiments in the laboratory. Chemical restraint may interfere with natural fly vector–mouse interactions and therefore potentially affect the outcome of transmission experiments. Conversely, manual restraint is labour-intensive and exposes laboratory animals to excessive restraining-related discomfort. We report development of a mouse restraining device (Infectra ® -kit) that allows essential transmission studies to be carried out with minimal human manipulation and without the need for anaesthesia. Infectra ® -kit can be used as a single unit for restraining one mouse or as eight-assembled units, thus significantly improving efficiency of a single operator in comparison to manual restraint. The kit was validated by comparing feeding success in tsetse flies fed on mice restrained using Infectra ® -kit (Group I) to those manually restrained (Group II). The mean ± SE % feeding success was 75.0 ± 8.2% and 82.1 ± 8.2% for tsetse flies in Groups I and II respectively. Statistical analysis using two sample t -test showed no significant difference between the two groups at p ≤ 0.05, indicating that Infectra ® -kit as a restraining device was as good as the conventional manual restraint method. The main benefits of using Infectra ® -kit for transmission studies therefore include reduction of man-hours and animal restraining-related discomfort. In addition, the risk of accidental injury to laboratory personnel by either mice or tsetse flies is minimized, which is an important consideration when working with zoonotic parasites.

Africa's Bane Tsetse are blood-feeding, fast-flying flies that transmit a range of Trypanosoma spp. protozoan pathogens, which cause sleeping sickness in humans and their nagana in their livestock. The International Glossina Genome Initiative (p. 380 ) sequenced the genome of Glossina morsitans and identified the genes for many attributes of the tsetse's remarkable biology, including viviparity and the expression of analogs of mammalian milk proteins. Tsetse are host to several specific symbionts that appear to synthesize essential nutrients for the fly and also to hitherto undiscovered parasitoid-derived viruses. Deeper exploration of this genome will reveal what makes these fly species so host- and trypanosome specific.

The level of the trypanocidal drug melarsoprol was determined in serum and cerebrospinal fluid (CSF) of six healthy vervet monkeys after intravenous application of the drug following a standard treatment schedule and a recently suggested alternative protocol. The maximum serum levels measured were about 3 μg/ml. A three-compartment model was used to analyze the serum data. The mean residence time calculated for melarsoprol in serum was 18 h, the volume of distribution was 3.6 l/kg and the clearance was 3.5 ml/min∗kg. In the CSF the drug levels were generally very low, not exceeding 55 ng/ml, and the adaptation of the drug levels was found to be very slow. The comparison of the drug concentrations required to eliminate trypanosomes in vitro and the drug concentrations reached in the CSF during treatment revealed that the latter might be insufficient in some cases to eliminate all trypanosomes from this site. The peak serum levels during alternative application of the drug were lower compared to those during empirical treatment. No evidence for drug cumulation in the body was found. The results of this study are compared with recent pharmacokinetic data from human patients, and discussed in the context of the problem of relapses and reactive encephalopathy occurring after treatment of sleeping sickness.