Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda.

Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. The HAT is caused by Trypanosoma brucei rhodesiense (Tbr) parasite in eastern and southern Africa, with suramin as drug of choice for treatment of early stage of the disease. Suramin treatment failures has been observed among HAT patients in Tbr foci in Uganda. In this study, we assessed Tbr parasite strains isolated from HAT patients responsive (Tbr EATRO-232) and non-responsive (Tbr EATRO-734) to suramin treatment in Busoga, Uganda for 1) putative role of suramin resistance in the treatment failure 2) correlation of suramin resistance with Tbr pathogenicity and 3) proteomic pathways underpinning the potential suramin resistance phenotype in vivo. We first assessed suramin response in each isolate by infecting male Swiss white mice followed by treatment using a series of suramin doses. We then assessed relative pathogenicity of the two Tbr isolates by assessing changes pathogenicity indices (prepatent period, survival and mortality). We finally isolated proteins from mice infected by the isolates, and assessed their proteomic profiles using mass spectrometry. We established putative resistance to 2.5 mg/kg suramin in the parasite Tbr EATRO-734. We established that Tbr EATRO-734 proliferated slower and has significantly enriched pathways associated with detoxification and metabolism of energy and drugs relative to Tbr EATRO-232. The Tbr EATRO-734 also has more abundantly expressed mitochondrion proteins and enzymes than Tbr EATRO-232. The suramin treatment failure may be linked to the relatively higher resistance to suramin in Tbr EATRO-734 than Tbr EATRO-232, among other host and parasite specific factors. However, the Tbr EATRO-734 appears to be less pathogenic than Tbr EATRO-232, as evidenced by its lower rate of parasitaemia. The Tbr EATRO-734 putatively surmount suramin challenges through induction of energy metabolism pathways. These cellular and molecular processes may be involved in suramin resistance in Tbr.

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model.

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n = 10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0-15, (b) acute: 16-30, (c) sub-acute: 31-45 and (d) chronic: 46-60 dpi. Other virulence biomarkers identified included: pre-patent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B), pentamidine, diminazene aceturate and suramin, using mice groups (n = 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. A cure rate of at least 80% was achieved for all test isolates with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr clones from a single HAT focus and confirms that this variations is not a significant determinant of isolate sensitivity to anti-trypanosomal drugs.

Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains.

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.

Comparative pathogenicity of Trypanosoma brucei rhodesiense strains in Swiss white mice and Mastomys natalensis rats.

We evaluated Mastomys natelensis rat as an animal model for Rhodesian sleeping sickness. Parasitaemia, clinical and pathological characteristics induced by T. b. rhodesiense isolates, KETRI 3439, 3622 and 3637 were compared in Mastomys rats and Swiss white mice. Each isolate was intra-peritonially injected in mice and rat groups (n=12) at 1×10(4) trypanosomes/0.2mL. Pre-patent period (PP) range for KETRI 3439 and KETRI 3622-groups was 3-6 days for mice and 4-5 days for rats while for KETRI 3637-infected mice and rats was 5-9 and 4-12 days, respectively. Pairwise comparison between PP of mice and rats separately infected with either isolate showed no significant difference (p>0.05). The PP's of KETRI 3637-infected mice were significantly (p>0.01) longer than those infected with KETRI 3439 or KETRI 3622, a trend also observed in rats. The second parasitaemic wave was more prominent in mice. Clinical signs included body weakness, dyspnoea, peri-orbital oedema and extreme emaciation which were more common in rats. Survival time for KETRI 3439 and 3622-infected groups was significantly (p<0.05) longer in mice than rats but similar in KETRI 3637-infected groups. Inflammatory lesions were more severe in rats than mice. All mice and KETRI 3622-infected rats had splenomegaly, organ congestion with rats additionally showing prominent lymphadenopathy. KETRI 3439-infected rats showed hemorrhagic pneumonia, enteritis with moderate splenomegaly and lymphadenopathy. KETRI 3637-infected rats had the most severe lesions characterized by prominent splenomegaly, lymphadenopathy, hepatomegaly, enlarged adrenal glands, organ congestion, generalized oedemas, gastroenteritis, pneumonia and brain congestion. KETRI 3637-infected Mastomys is a suitable model for studying pathophysiology of HAT.

Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts.

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 1parasite/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.

Infectra(®)-kit: a device for restraining mice and confining tsetse flies during trypanosome infection transmission experiments.

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.

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates.

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.