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.

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.