Ethics
Approval for performing our experiments on mice was obtained from the Kenya Agricultural and Livestock Research Organization -Biotechnology Research Institute - (KALRO- BioRI) Review Board (C/Biori/4/325/II/49) and the protocol reviewed by and approved by the BioRI-KALRO Institutional Animal Care and Use Committee (IACUC).
Selection Of Trypanosomes Isolates
Five Tbr isolates were used in this study (Table 1). They were selected from BioRI (formerly KETRI) cryo-bank and were originally isolated from HAT patients in the three east African countries [14].The isolates had gone between1-8 eight passages in mice. Old and recent isolates were selected for this study.
Table 1
Isolates of T.b. rhodesiense collected from different parts of eastern Africa
Stabilate No: | Locality | Year of isolation | Host of isolation | Passage No | |
KETRI 3738 (2537) | Banda, Busoga, Uganda | 1972 | Human | 8 | |
KETRI 3537 | Bungoma, Western Province, Kenya | 1998 | Human | 3 | |
KETRI 2656 KETRI 3459 EATRO 2291 | Lambwe valley,Kenya Kitanga, Tanzania Busoga, Uganda | 1983 1960 1976 | Human Human Human | 2 3 1 | |
Molecular Characterization Of Trypanosome Isolates
The parasites (isolates) were originally ascribed as Tbr based on their host identity (human), clinical manifestation and their appearance under microscopy (as trypanosomes). We therefore validated their Tbr species status via PCR. We separately extracted and purified DNA from the individual isolates using Qiagen DNA easy blood and tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. We then separately performed PCR amplification targeting Tbr species-specific SRA gene[15] in 10∝ l reactions consisting of 2.5 µl DNA, 0.125 units of Taq polymerase (Promega, CA, USA) 1X PCR buffer, 0.2 mM dNTPS, 2 mM MgCl2, 1 ∝M each of forward (SRA A) and reverse (SRA E) primers. The thermocycling conditions included initial step 95oC for 3 min, followed by 35 cycles of 95oC for 30 s, 60oC for 30 s, 72OC for 1 min and final extension at 720C for 2 min. We included DNA from a validated Tbr as a positive control KETRI 2537 (KETRI 3738)[16] and PCR water as well as T.b.brucei as negative control. We resolved the amplicons on 2% molecular grade Top vision agarose (Thermo Scientific) stained with ethidium bromide, and documented the gel using UVITEC (Cambridge) gel imager.
Experimental Animals
We obtained 145 male Swiss White mice weighing 25–30 g from KALRO-BioRI Small Animals Breeding Unit. The mice were housed in groups of 10 in standard mouse cages and using woodcarvings as bedding material and maintained on a diet consisting of commercial pellets (Unga® Kenya Ltd) and water ad libitum. The mice were kept in a locked room under natural light. Room temperature and humidity were not regulated. We acclimatized the mice to experimental room conditions for two weeks and dewormed them using Noromectin®, Norbrook, UK at 0.2 mg/kg as described by [17] to ensure that they were free of ecto- and endo-parasites. We obtained during this acclimatization period base line data on packed cell volume (PCV) and body weight prior to infection.
Experimental Design
Four (4) donor mice per isolate were immunosuppressed using cyclophosphamide 300 mg/kg for three consecutive days[18] after which the recovered cryopreserved T.b.rhodesiense isolates shown in Table 1were injected for multiplication.The mice were monitored for parasitaemia for 21 days post infection, a period known to be sufficient for development of late stage disease in the mouse model [19]. They were then euthanized using concentrated carbon dioxide (CO2). At least one mL of heart blood trypanosomes were collected in 5 µl of 10% EDTA enough to prevent coagulation and pooled from the four donor’s mice while brain tissues from the four donor mice were also pooled and suspended in cold PSG pH 8.0. Brain tissues were washed in 3 Ml PSG pH 8.0 buffer per wash for at least ten times (total 30Ml PSG buffer) and presence of trypanosomes in every wash carefully examined under the microscope. When no trypanosome could be detected, the final buffer wash was discarded, the brain excised using sharp pair of scissors and gently homogenized in PSG pH 8.0. The supernatant containing brain trypanosomes (CNS) from the four donor mice was pooled. Slide smears of CNS trypanosomes of each of the isolates were made from the brain homogenates. Similarly, smears of BSF trypanosomes from each of the Tbr isolates were made from pooled heart blood. The density of trypanosomes in the pooled supernatant collected from the brain homogenates (Table 3) was then quantified using a haemocytometer. Similarly, the density of the BSF trypanosomes in the pooled heart blood (Table 3) were also quantified and adjusted to the inoculum level of the supernatant containing CNS form trypanosomes using PSG pH 8.0 (Table 3). Either BSF or CNS form of trypanosomes (BSF and CNS) were then intraperitoneally injected into ten experimental mice while five non-infected mice acted as control. The infected mice were monitored for the following parameters: pre-patent period (PP), parasitaemia progression, PCV and body weight once a week, survival, gross pathology, and histopathology. At 30 days post infection, four mice from each group including two non-infected control were sacrificed for gross lesions (lesions and organ weights) and histopathology. The remaining mice were humanly euthanized and disposed.
Pre-patent period (pp) and parasitaemia progression
Blood for estimation of parasitaemia levels was collected daily from each mouse using the tail tip amputation method [20]. The pre-patent period and parasitaemia progression were determined using the rapid matching method [21]. The infected mice were monitored for 30 DPI. The end point of the infected mice was determined by observation of clinical signs such as lethargy and hackle hair, as well as PCV drop of approximately 25% with consistent high parasitaemia levels of 1 × 109/mL − 1 for at least three consecutive days. The animals were sacrificed immediately by CO2 asphyxiation in accordance with guidelines of the Institutional Animal Care and Use Committee (IUCAC) as described by [22] and recorded as dead animals.
Determination Of Morphology Of Trypanosomes
A drop of blood from mice infected with BSF or CNS derived trypanosomes was collected as infection progressed. Thin smears were then prepared and examined using Leica DM500 microscope at high magnification with oil immersion objective (10 × 100). The length of the trypanosome was measured from the posterior end to the anterior end including the free flagellum as previously outlined by Stephen [23] On average, 50 trypanosomes of BSF or CNS were measured at every time point (DPI) per isolate.
Packed cell volume (PCV) and body weight
These were measured once a week as outlined by Naessens [24] for PCV and by Gitonga [25] for body weight.
Survival
All control mice survived up to the end of the experimental period of 30 days and their survival time data were therefore categorised as censored. The shortest survival time was observed in mice infected with EATRO 2291 at 13.7 ± 2.0 and 12.7 ± 1.5 for BSF or CNS infected mice, respectively (Fig. 7 (v). This was followed by mice infected with KETRI 2656 at 17.6 ± 1.2 and 15.9 ± 0.3 for BSF or CNS infected mice, respectively (Fig. 7 (iii). Mice infected with KETRI 3738, KETRI 3537 and KETRI 3459 survived for period between 26 DPI and end of experiment (Fig. 7 (i), (ii) and (iv)). The p values associated with Wilcoxon and Logrank tests of homogeneity for the BSF or CNS forms of individual isolate ranged between 0.1 to 0.5 and 0.1 to 0.3 respectively showing no significant difference between the groups both at early and longer survival times. However, when the two groups are compared, CNS infected mice survival was shorter compared to BSF infected mice, although not significantly different (p > 0.05) (Fig. 7 (vi).
Gross Pathology And Histopathology
Four infected mice from each of the BSF or CNS derived trypanosomes groups including two control mice were sacrificed at 31 DPI for KETRI 3537 and 3738, KETRI 3439 at 29 DPI, EATRO 2291 at 15 DPI and the only surviving mouse of KETRI 2656 at 18 DPI. The body weight of each mouse was determined after which the mouse was euthanized, dissected and the brain, spleen, kidneys, liver, lungs and heart collected and weighed using a weighing balance (Mettler Toledo PB 302 ®, Switzerland). These organs were preserved in 10% formalin and thereafter processed for histopathology as previously described [27]..
Statistical analysis
Analysis was done to test the significant differences between the BSF and CNS forms per isolates in PP, parasitaemia progression, PCV, body weights, trypanosomes length and survival in the two groups. The data obtained from the study were summarized as means ± standard error of mean, while the differences between and within the means were analyzed using one-way ANOVA. The analyses were conducted using GenStat, UK where p ⩽ 0.05 were considered statistically significant. General Linear Model in SAS Release 8.02 was used to analyze data on the length of the trypanosome. Differences between any two means were considered significant at p < 0.05. Survival data analysis was carried out employing the Kaplan–Meier method on StatView (SAS Institute, Version 5.0.1) statistical package for determination of survival distribution function. Rank tests of homogeneity were used to determine the effect on host survival time of BSF- and CNS-infected mice [29].