All Experimental protocol were approved and conducted with strict adherence to guidelines of the institutional animal care and use committee of the University of Jos Plateau state, Nigeria, which are in accordance with the Principle of Laboratory Animal Care Of the Canadian Council on Animal Care Guide (CACC) 2^nd edited vol 1 1993.

A total of 20 rats and 20 mice were used for the experiment. The animals were obtained from the Animal experimental unit Department of Pharmacology, Faculty of Pharmaceutical Sciences University of Jos. They Animals were allowed to acclimatise for approximately 2 weeks in the Animal house of the Faculty of Pharmaceutical Sciences, University of Jos, Nigeria. The rats and mice were placed under standard hygienic conditions in plastic cages with steal cover. Wood shavings were used as bedding which were changed every week. They were fed ad libitum with standard rodent feeds.

Trypanosoma brucei brucei used for the study was obtained from Nigerian institute for Trypanosomaisis Research Vom Plateau state, Nigeria. The parasite was maintained by serial passage in donor rats. Parasitaemia was maintained daily by preparing wet mount and viewing under a light microscope at 40×.

A total of forty rodents (20 rats and 20 mice) were used (Figure 1). The Mice and Rats were grouped base on the age group of 1 - 5 weeks and 8 - 15 weeks with each age group comprising of 10 mice and 10 rats and were further sub-divided into 2 groups of 5 each. The first group of rodents each comprised of younger age group mice and rats had 2 males: 2 Females and 1 control were inoculated orally with the parasite strain Trypanosoma brucei brucei. Whereas the second group which also comprised of 2 males: 2 Females and 1 control were however inoculated with the parasite intraperitoneally. The older rodents (7 - 15 weeks) of a group of 10 were also sub-divided into 2 groups of 5 each. The older rodents were also inoculated in the fashion of the younger ones i.e. orally and intraperitonealy with the parasite strain Trypanosoma brucei brucei parasite. Trypanocidal drugs (Isometamidium chloride) were administered to rodents that have confirmed established parasitaemia (particularly those infected intraperitonealy).

Blood films were made from the caudal vein of each rat after sterilization. Trypanosome count was determined by examination of the wet mount microscopically

at ×40 magnification using the “Rapid Matching” method. Briefly, this method involves microscopic counting of parasites per field in pure blood or blood appropriately diluted with PBS (pH 7.2).

Inoculum was prepared from infected whole blood collected from infected or donor rat. Blood sample was collected from donor rat when parasitaemia was between 20% to 30% in the blood. The total volume obtained separated into two each in an anti-coagulant tube. Blood smear was prepared in order to estimate the number of Trypanosomes per millilitre of blood present in each dose of inoculum.

Phosphate Buffer Saline (PBS) was used to dilute the oral inoculum. Phosphate Buffer Saline served as a neutraliser to the high acidic content of the stomach of the mice and rats respectively. Blood sample was collected from a donor passaging rat and put into an anti-coagulant tube. The blood was rinsed three times by centrifuging at 6000 revolutions per minutes (rpm) by discharging the supernatant and adding the Phosphate Buffer Saline. At the last washing step infected Red Blood Cells was gently Re-suspended with a specific volume of Phosphate Buffer Saline so as to produce an approximate concentration of 1 × 10⁸ Red blood Cells per inoculum. The volume of blood and PBS needed to create a concentration of approximately 1 × 10⁸ red blood cells per inoculum was calculated using the formula:

RBC = Average RBC × Estimated Parasitaemia 100 [7]

Oral Route of Inoculation

Oral Inoculation was achieved with the use of a cannula inoculums gently deposited into the oral cavity and animal were allowed to swallow. This method was preferred so as to avoid any potential damage to the oral epithelium the gavage could cause. Controls were inoculated with stride Phosphate Buffer Solution (PBS).

Weighing of the animals was done using electronic weighing balance. These were carried out 3 times with the first taken two weeks before inoculation; the second was taken 3 days after pre-patent period of infection and the last 3 days after treatment.

The Pack Cell Volume Count was determined using the micro-haematocrit reader. Blood samples were obtained from the tail of both rodents using a capillary tube and then transferred to the micro-haematocrit centrifuge and then spinned at 15,000 revolutions per minute. Measurements were carried out 3 times with the first taken two weeks before inoculation; the second was taken 3 days after pre-patent period of infection and the last 3 days after treatment.

Thick and thin films were prepared from each subject’s blood sample. The thin film was fixed with absolute methanol and both thick and thin films were stained with Leishman’s stain and were then examined microscopically with oil immersion (×100) objective. The parasite counting was done using the thick blood film and the thin blood films on wet mounts in accordance to the method describe by WHO [8] [9] [10]. The parasite counts in relation to Red Blood Cell count were converted to parasite per microliter of blood using mathematical formula:

Parasitaemia ( μl ) = No . Of parasite 2 00 × 8 000

where 8000 = putative means of Red Blood Cell.

The numbers of parasites were counted against 200 Red Blood Cell using laboratory counter (N.B. Once started, a field is always counted to the end; therefore, it is usual that the final red blood count will be over 200).

Isomethamedium chloride was our drug of choice. Trypanocidal drug treatments were given for those inoculated intraperitoneally after observation of peak parasitaemia by wet film examination [11]. Drug injections were given intraperitonealy with each rodent receiving Isomethamedium chloride at a dose of 4 mg/kg base on their body weight.

The initial mean weight of the younger rats taken 2 weeks before inoculation was at 39 g; 4 days after inoculation was at 62.13 g and 2 days after treatment was at 104.65 g. The statistical analysis before and after inoculation showed there was a significant difference (P < 0.05) and this also applies to after inoculation and after treatments where there was a significant difference (P < 0.05) as seen in Table 1. Similarly, the initial mean weight of the older rats which was taken 2 weeks before inoculation was at 111.43 g; four (4) days after inoculation was at 132 g and 2 days after treatment was at 104.65 g. The statistics analysis indicates that before and after inoculation there was a significant difference (P < 0.05) and this also applies to after inoculation and after treatments where there was a significant change (P < 0.05) as seen on Table 1 based on one way ANOVA.

Table 1 also shows the mean weight (g) for younger rats inoculated orally having an initial mean weight taken 2 weeks before inoculation at 40.16 g; four (4) days after inoculation at 68.16 g and 2 days after treatment at 68.06 g. The statistics analysis indicates that before and after inoculation there was a significant difference (P < 0.05); but after inoculation and after treatments there was no significant difference at P > 0.05. The same pattern was observed between the older rats’ age group with an initial weight of 112.71 g taken 2 weeks before inoculation and 137.05 g taken 4 days after inoculation and 137.4 g 2 days after treatment. Though the statistics indicates that before and after inoculation there was a significant difference (P < 0.05); but after inoculation and after treatments there was no significant difference (P > 0.05) based on one way ANOVA.

Comparative study based on inoculation and treatment as seen in Table 1 indicates that the weight of both younger and older rats inoculated orally were higher in weight than those inoculated intraperitoneally. Statistical analysis using ANOVA to compare routes of inoculation indicate that P < 0.05 at 95% confidence interval. In all, fewer deaths were recorded among the rats especially among the younger female rats (1 - 5 weeks) as compared to their male counterpart.

Table 1 shows the mean weight (g) for younger mice inoculated intraperitoneally having an initial mean weight taken 2 weeks before inoculation at 20.20 g; 4 days after inoculation was at 24.20 g and 2 days after treatment at 14.86 g. The statistics analysis using ANOVA indicates that before and after inoculation there was a significant difference at P < 0.05; but after inoculation and after treatments there was a significant difference (P < 0.05). Similarly, Table 1 shows the mean weight (g) for younger mice inoculated orally having an initial mean weight taken 2 weeks before inoculation at 20.6 g; 4 days after inoculation at 23.18 g and 2 days after treatment at 19.3 g. The statistical analysis using one way ANOVA indicates that before and after inoculation there was a significant difference at P < 0.05; but after inoculation and after treatments, there was no significant difference at P > 0.05.

Table 1 also shows the mean weight (g) for older mice inoculated intraperitoneally having an initial mean weight taken 2 weeks before inoculation at 23.36 g; 4 days after inoculation at 26.85 g and 2 days after treatment at 14.46 g. The statistics analysis using one way ANOVA indicates that before and after inoculation there was a significant difference (P < 0.05), and after inoculation and after treatments there was also an observed significant difference at P < 0.05. Similarly, Table 1 shows the mean weight (g) for older mice inoculated orally having an initial mean weight taken 2 weeks before inoculation at 24.26 g; 4 days after inoculation at 31.00 g and 2 days after treatment at 30.86 g. The statistics analysis using one way ANOVA indicates that before and after inoculation there was a significant difference at P < 0.05; but after inoculation and after treatments there was no significant difference at P > 0.05.