The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study.

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense ( Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies.

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.

Route of inoculation influences Trypanosoma congolense and Trypanosoma brucei brucei virulence in Swiss white mice.

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.

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.

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.

Effects of Ethidium (homidium bromide) on female reproductive performance of laboratory-reared tsetse flies, Glossina morsitans morsitans Westwood (Diptera: Glossinidae).

Ethidium (homidium bromide) is a trypanocide likely to be encountered as a violative residue in blood collected from abattoirs for feeding laboratory tsetse flies. We investigated its effect on female reproduction of Glossina morsitans morsitans. One-milligram homidium per kilogram body weight was intra-muscularly administered to four steers and blood aseptically collected from them between 15 and 30 min post-treatment, analysed for homidium levels and processed for tsetse feeding. Two hundred teneral female flies were fed on homidium-treated diet while a control group of similar number was given untreated diet and the reproductive performance of the two groups statistically compared. Ethidium, at 266.1 ng homidium/ml blood diet, halved A-class portion of F(1)-pupae, highly reduced decline of F(1)-progeny quality associated with aging parents, but had no significant effect on the pupae viability, fecundity and abortion rate of the flies. We therefore concluded that Ethidium has beneficial effect on laboratory tsetse attributable to clearance of unfavourable microbes mediated by the drug, and could be used as a tsetse diet additive.