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

Acta Tropica 150 (2015) 23–28

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Comparative pathogenicity of Trypanosoma brucei rhodesiense strains in Swiss white mice and Mastomys natalensis rats Margaret Wanjiku Muchiri a,b , Kariuki Ndung’u a,∗ , James Karuku Kibugu a , John Kibuthu Thuita a , Purity Kaari Gitonga a , Geoffrey Njuguna Ngae c , Raymond Ellie Mdachi a , John Maina Kagira b a

Kenya Agricultural and Livestock Research Organization (KALRO), Biotechnology Research Institute (BioRI), P. O. Box 362, Kikuyu, Kenya Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000–00200, Kenya c Kenya Food Crop Research Institute, P. O. Box 30148, Nairobi, Kenya b

a r t i c l e

i n f o

Article history: Received 28 March 2015 Received in revised form 8 June 2015 Accepted 12 June 2015 Available online 19 June 2015 Keywords: Mastomys rats Rhodesian HAT rat model Mice

a b s t r a c t 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 × 104 trypanosomes/0.2 mL. 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 3637infected Mastomys is a suitable model for studying pathophysiology of HAT. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Sleeping sickness also referred to as human African trypanosomiasis (HAT) is of great medical concern in sub-Saharan Africa where more than 66 million people are at risk of infection; HAT is also an obstacle to rural development (Brun et al., 2010; World Health Organization, 2004). The disease is caused by two trypanosome species, Trypanosoma brucei rhodesiense and T. b. gambiense, which are cyclically transmitted by tsetse flies (World Health Organization, 2004). T. b. rhodesiense causes an acute zoonotic disease restricted to eastern and southern Africa while the more chronic T. b. gambiense-HAT is found in central and

∗ Corresponding author. E-mail address: [email protected] (K. Ndung’u). http://dx.doi.org/10.1016/j.actatropica.2015.06.010 0001-706X/© 2015 Elsevier B.V. All rights reserved.

western Africa (Brun et al., 2010; World Health Organization, 2004; Mulligan, 1970). HAT is classified into early (first or haemolymphatic) and late (second) stage disease; the late stage is sometimes further divided into transitional phase with parasites in cerebrospinal fluid without central nervous system (CNS) involvement and meningoencephalitic phase where CNS is affected (World Health Organization, 2004; Burundi et al., 1995). The drugs used for curative treatment of T. b. rhodesiense infections are suramin and melarsoprol for the early and late stages of the disease respectively (Brun et al., 2010; Nok, 2003). Emergence of drug resistance in sleeping sickness (Kagira and Maina, 2007; Nok, 2003), trypanocidal drug failure, lack of incentive for development of new trypanocides and unavailability of anti-trypanosomal vaccines (Geerts and Holmes, 1998; Gutterridge and Coombs, 1977) are major impediments to effective management of Rhodesian sleeping sickness.

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Murine models of HAT have been developed and used in research involving disease pathogenesis (Kibugu et al., 2009; Jennings et al., 1977), preclinical efficacy trials of novel drugs and evaluation of drug regimens (Thuita et al., 2008; Gichuki and Brun, 1999; Ndung’u and Akol, 1988), and validation of scientific innovations (Ndung’u et al., 2013). The development of these has been influenced by the variable infectivity patterns exhibited by trypanosomes. T. b. rhodesiense (EATRO 1989) induces a chronic infection in mice (Fink and Schmidt, 1979) and its derivative, KETRI 2537 has therefore been used to develop a mouse model of late stage HAT (Kagira et al., 2007; Gichuki and Brun, 1999). However, most T. brucei parasites cause acute disease in mice and cannot be used to develop late stage disease model (Morrison et al., 1978), therefore the pathogenicity of newly isolated strains has to be evaluated to ascertain their suitability for development of animal models. Murine models are preferred due to ease of handling and reduced cost of experiment such as product evaluations compared with models utilizing larger mammals (Ndung’u et al., 2013; Peregrine, 1994). The utility of murine models is however limited by small circulating blood volume available for sampling risking animal exsanguination (Kibugu et al., 2010). This is particularly a concern in pathogenesis studies requiring frequent bleeding. Mastomys rats, commonly used in studies involving T. b. gambiense (Farah et al., 2014) have relatively larger circulating blood volume than mice. However its suitability as an animal model for T. b. rhodesiense infection has not been investigated. The present study was therefore designed to: (i) elucidate the pathogenicity of three uncharacterized T. b. rhodesiense parasites in order to determine their suitability for development of late-stage rodent models (ii) evaluate the suitability of Mastomys natalensis rats as an animal model for T. b. rhodesiense by comparing parasitological and pathological changes in the rats and mice-infected with the T. b. rhodesiense parasites.

improved chamber then diluted using PSG pH 8.0 to 1 × 104 trypanosomes/0.2 mL and used to infect the experimental mice and rats intraperitonially (ip). 2.3. Experimental design To infect mice and rats, donor mice were euthanized at peak parasitaemia and blood harvested as described earlier (Kagira et al., 2007). Three groups of 12 mice were infected with KETRI 3439 (Group A), KETRI 3622 (Group B) and KETRI 3637 (Group C) trypanosomes at 1 × 104 trypanosomes/0.2 mL per mouse (Gichuki and Brun, 1999; Kagira et al., 2007) and monitored for 55 days. The pre-patent period (PP), parasitaemia development, survival times, clinical signs and gross pathology were recorded as previously described (Kibugu et al., 2009). This was repeated with 3 groups (Groups D, E and F, respectively) of Mastomys natalensis rats. 2.4. Monitoring of parasitaemia and clinical changes in mice and rats

2. Materials and methods

Blood of the infected experimental animals was collected daily for 55 days by tail snip method and examined by light microscopy to detect trypanosomes. The matching technique described by (Herbert and Lumsden, 1976) was employed to score parasitaemia levels. The pre-patent period (PP), for each infected animal was determined and recorded. Survival times for each animal were monitored for 55 days post-trypanosome infection. This time was based on maximum survival time of 54–65 days observed in T. b. rhodesiense-infected laboratory rodents (Kibugu et al., 2009; Fink and Schmidt, 1979). For animals surviving beyond this period, the survival time was recorded as 55 days and categorized as censored data. Clinical status of the animals was monitored daily as described by (Gichuki and Brun, 1999). The sick animals were euthanised when they were in extremis and gross pathology examination performed.

2.1. Ethics

2.5. Data analysis

All protocols and procedures used in this study involving laboratory animals were reviewed and approved by the KALRO-BioRI Institutional Animal Care and Use Committee.

Pre-patent period data were subjected to analysis of variance and mean separation on Genstat statistical package (Genstat 5 Release 3.2 Lawes Agricultural Trust, IACR-Rothamsted). 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 (i) each trypanosome isolate on mice and rats, (ii) the 3 trypanosome isolates separately on mice and rats (Everitt and Der, 1998).

2.2. Materials 2.2.1. Animals Thirty six (36) male White Swiss mice (25–30 g body weight) and 36 male Mastomys natalensis rats (100–150 g body weight) were obtained from Kenya Medical Research Institute (KEMRI) and KALRO-BioRI Small Animal breeding units respectively. The animals were maintained on mice pellets (Unga Feeds Ltd., Kenya) and water ad libitum at room temperature, and wood-chippings provided as bedding material. They were acclimatized for 7 days before the experiment commenced. 2.2.2. Trypanosomes Three cryo-preserved T. b. rhodesiense stabilates designated KETRI 3439, KETRI 3622 and KETRI 3637 which were previously isolated from human hosts at Busia, Kenya in 1997, 1998 and 1999, respectively, were obtained from the KALRO-BioRI Trypanosome bank. They were suspended in cold phosphate saline glucose (PSG), pH 8.0 and expanded in irradiated donor mice as described by (Gichuki and Brun 1999). At the exponential phase of parasitaemia development, one donor mouse was euthanized, bled from the heart into EDTA tubes and parasitaemia quantified using Neubauer

3. Results 3.1. Parasitaemia development The pre-patent period (PP) of KETRI 3439, KETRI 3622 and KETRI 3637-infected mice (Groups A–C) and rats (Groups D–F) are shown in Table 1 while their parasitaemia patterns are given in Fig. 1. The mean PP’s of the animal groups infected with KETRI 3637 were significantly longer (p < 0.01) compared with those infected with KETRI 3439 or KETRI 3622 (Table 1). It was also observed that there was no significant difference in pre-patent periods between mice and rats infected with the three trypanosomes. Further, the three trypanosome isolates showed highly significant inter-group variation (p < 0.01) in pre-patent periods. The parasitaemia was characterized by two prominent wave in mice and three waves in

M.W. Muchiri et al. / Acta Tropica 150 (2015) 23–28

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Table 1 Pre-patent periods in mice and rats infected with different T. b. rhodesiense stabilates expressed in days (Mean ± SE and range). Animal species

KETRI 3439

KETRI 3622

Mean ± SE Rats Mice a

Range

4.58 ± 0.15 4.00 ± 0.37

4–5 3–6

KETRI 3637

Mean ± SE

Range

Mean ± SE

Range

4.25 ± 0.13 4.83 ± 0.24

4–5 3–6

6.08a ± 0.81 6.67a ± 0.33

4–12 5–9

PP’s of animals infected with KETRI 3637 were significantly longer than those infected with other two trypanosomes.

Parasitaemia (antilog)

rats (Fig. 1). The initial peak occurred during the first week post infection at 3–12 days for KETRI 3439-infected mice; 4–10 days for KETRI 3622-infected mice; 5–13 days for KETRI 3637-infected mice and 4–12 days for KETRI 3439-infected rats; 4–9 days for KETRI 3622-infected rats; 4–10 days for KETRI 3637 rats and thereafter parasitaemia progressed as shown in Fig. 1. The subsequent parasitaemia waves which persisted until death, were bigger in mice than in rats. For all the three trypanosome isolates, the second wave parasitaemia was higher in mice than in rats (Fig. 1(i)–(iii)).

3.2. Clinical changes and host survival At extremis the clinical signs observed in trypanosome-infected mice (Groups A–C) and rats (Groups D–F) respectively, infected with the three trypanosomes included general body weakness, dyspnoea, peri-orbital oedema and extreme emaciation. Generally, these clinical signs were more common in rats than mice. Fig. 2 shows the survival distribution functions of mice and rats infected with the three trypanosome isolates, KETRI 3439, KETRI

10 8 6 4 2 0 1

3

5

7

9

11

13

15

17

19

21

23

25

Time post-infection (days) KETRI 3439-infected mice

KETRI 3439-infected rats

Parasitaemia (Antilog)

(i): KETRI 3439-infected mice (Gp. A) and rats (Gp. D) 10 8 6 4 2 0 1

3

5

7

9

11

13

15

17

19

21

23

25

27

29

31

33

35

Time post-infection (days) KETRI 3622-infected mice

KETRI 3622-infected rats

(ii): KETRI 3622-infected mice (Gp. B) and rats (Gp. E) Parasitaemia (Antilog)

10 8 6 4 2 0 1

3

5

7

9

11

13

15

17

19

21

23

25

27

29

31

33

35

Time post-infection (days) KETRI 3637-infected mice

KETRI 3637-infected rats

(iii): KETRI 3637-infected mice (Gp.C) and rats (Gp. F) the figure title Fig. 1. Parasitaemia patterns in mice and rats infected with Trypanosoma brucei rhodesiense isolates.

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M.W. Muchiri et al. / Acta Tropica 150 (2015) 23–28 1

1

.8

.8 Cum. Survival

Cum. Survival

.6

.6 .4

.4

.2

.2 0

0 0

10

20

30 Time

40

50

0

60

10

Event Times (Mice-KETRI 3439)

Cum. Survival (Mice-KETRI 3439) Cum. Survival (Rat-KETRI 3439)

20

Time

30

40

Cum. Survival (Mice-KETRI 3622) Cum. Survival (Rat-KETRI 3622)

Event Times (Rat-KETRI 3439)

Censor Times (Mice-KETRI 3622)

(i): Mice and rats in fected with KETRI 3439

60

Event Times (Rat-KETRI 3622) Censor Times (Rat-KETRI 3622)

(ii): Mice and rats infected with KETRI 3622 1

1

.8

.8

Cum. Survival

50

Event Times (Mice-KETRI 3622)

.6

.6 Cum. Survival

.4

.4 .2

.2 0

0 0

10

20

30 Time

Cum. Survival (Mice-KETRI 3637) Cum. Survival (Rat-KETRI 363) Censor Times (Mice-KETRI 3637)

40

50

60

0

10

Event Times (Mice-KETRI 3637) Event Times (Rat-KETRI 3637) Censor Times (Rat-KETRI 3637)

(iii) Mice and rats infected with KETRI 3637

20

30 Time

40

50

Cum. Survival (Mice-KETRI 3439)

Event Times (Mice-KETRI 3439)

Cum. Survival (Mice-KETRI 3622)

Event Times (Mice-KETRI 3622)

Cum. Survival (Mice-KETRI 3637)

Event Times (Mice-KETRI 3637)

Censor Times (Mice-KETRI 3439)

60

Censor Times (Mice-KETRI 3622)

Censor Times (Mice-KETRI 3637)

(iv) Trypanosome-infected mice

1 .8 .6 Cum. Survival

.4 .2 0 0

10

20

30 Time

40

50

Cum. Survival (Rat-KETRI 3439)

Event Times (Rat-KETRI 3439)

Cum. Survival (Rat-KETRI 3622)

Event Times (Rat-KETRI 3622)

Cum. Survival (Rat-KETRI 3637)

Event Times (Rat-KETRI 3637)

Censor Times (Rat-KETRI 3439)

60

Censor Times (Rat-KETRI 3622)

Censor Times (Rat-KETRI 3637)

(v):Trypanosome-infected rats Fig. 2. Survival distribution functions of trypanosome-infected mice and rats.

3622 and KETRI 3637. The comparisons of infected animal species and infecting trypanosome on host survival are given in Fig. 2. The survival time for KETRI 3439-infected groups ranged from 30 to 54 days for mice compared to 10–27 days for the rats (Fig. 2(i)). The rank tests of homogeneity showed that Log Rank and Wilcoxon tests’ p-values were both highly significant (<0.0001). This indicated that the two groups differed at both early and larger survival times with significantly (p < 0.05) shorter survival times in rats (Gp. D) compared to mice (Gp. A) (Fig. 2(i)). For KETRI 3622-infected groups, survival time ranged from 34 to 53 days for mice (Gp. B) compared to 21–45 days in rats (Gp. E) (Fig. 2(ii)). The Wilcoxon test (p = 0.002) was both significant and less than of Log Rank test (0.028). Hence the two groups differed mainly at early survival times with significantly (p < 0.05) shorter early survival times in the rats (Gp. E) compared to mice (Gp. B) (Fig. 2(ii)). In the KETRI 3637-infected groups, survival time ranged from 28 to 36 days for mice (Gp. C) compared to 19–35 days for rats (Gp. F) (Fig. 2(iii)). The p-value associated with Log Rank test was not significant (p > 0.05) while that for the Wilcoxon test was not computed by the statistical program. This signifies that the two groups did not experience death events during the early phase of the infection and that there was no evidence of significant difference in larger survival times in rats (Gp. F) and mice (Gp. C) infected with KETRI 3637 (Fig. 2(iii)).

The survival of three mice groups each infected with one trypanosome isolate is compared in Fig. 2(iv) while the same for three rat groups is given in Fig. 2(v). The p-values associated with Log Rank and Wilcoxon rank tests were equal and significant (p < 0.0001) indicating evidence of significant difference in survival among the mice (Gps A–C) (Fig. 2(iv)) and rat (Gps D–F) (Fig. 2(v)) groups infected with KETRI 3439, KETRI 3622 and KETRI 3637 trypanosomes. Least survival times were observed in the KETRI 3637-infected group in mice (Fig. 2(iv)) and KETRI 3439-infected group in rats group (Fig. 2(v)).

3.3. Clinical pathological changes Gross pathological lesions observed in the trypanosomeinfected mice (Groups A–C) included enlarged spleen and congestion in most organs. There were no differences in severity of the lesions induced by the three trypanosome isolates in mice. In rats, differences in severity and organ involvement were observed in the gross pathology lesions induced by the trypanosome isolates. KETRI 3439-infected rats (Group D): The carcasses were mainly emaciated. Mild changes included petechial hemorrhages and congestion in the lungs (hemorrhagic pneumonia), mild congestion at

M.W. Muchiri et al. / Acta Tropica 150 (2015) 23–28

the pyrollic junction, and inflammation of the intestinal mucosa. Spleen and lymphnodes were moderately enlarged. KETRI 3622-infected rats (Group E): The gross pathology lesions were similar to those of the trypanosome-mice (Groups A–C) the prominent features being splenomegaly and congestion in most organs. The lymph nodes were also prominently enlarged. KETRI 3637-infected rats (Group F): Severe pathology lesions were observed in this group. The spleen was enlarged five-fold the normal size. Other enlarged organs were lymph nodes, liver and the adrenal glands. Lymphadenopathy was a prominent feature in this group. Most of the other organs were severely congested. The lung texture was consolidated (firm and brown–reddish) and on cutting the surface, a lot of foam was observed suggesting pneumonia. A lot of exudation was observed in most body cavities and manifested as ascites, hydropericardium and hydrocoele. Severe gastroenteritis characterized by multiple bleeding and healing ulcers (in the stomach), bloody ingested (food content) in stomach and intestines, and dark faeces (digested blood) in the large intestines. Some rats had watery mucoid contents and inflamed intestines (catarrhal enteritis). Congestion was observed in the brain meninges (outer layer) in some rats.

4. Discussion In the present study, we demonstrated the suitability of Mastomys natalensis rat as an animal model for pathogenesis studies of T. b. rhodesiense infection. Comparison of parasitaemic patterns suggested that the second parasitaemic wave was more prominent in mice than in rats infected with T. b. rhodesiense. This agrees with (Morrison et al., 1978) who observed differences in infectivity of the same trypanosome in different rodent hosts. Further, it is important to note that parasitaemia levels were generally higher in mice compared to rats in the present study. Indeed, extremely high parasitaemia levels is one of the limitations of the mouse models of trypanosomiasis (Magez and Caljon, 2011). This agrees with earlier studies using EATRO 1989 and KETRI 3741 (Kibugu et al., 2009; Fink and Schmidt, 1979), the three trypanosome isolates used in the present study produced chronic infection in mice. The present study further indicates that the pre-patent periods (PP) of mice and rats infected with KETRI 3637 were significantly longer than those infected with the other two isolates. (Sacks et al., 1980) found that the longer the PP, the less virulent is the trypanosome infection. Our finding is important because it has identified other T. b. rhodesiense trypanosomes to be used alongside EATRO 1989 and KETRI 3741 trypanosomes in late stage studies. Most T. brucei parasites cause acute disease in laboratory rodents making them unsuitable as meningoencephalitic phase late stage disease model. Also, meningoencephalitis is rare in T. b. rhodesiense-mice models (Fink and Schmidt, 1979; Keita et al., 1997). This necessitates development of a cheap and reliable novel small rodent disease model for late stage trypanosomiasis. Such experimental model will facilitate our understanding of the disease and evaluation of suitable drug regime for curing the late stages of disease (Gichuki and Brun, 1999). Development of such a model has been difficult because most T. brucei organisms used tend to cause acute rather than a chronic disease in experimental animals. Our present findings suggest KETRI 3637 infection in Mastomys natalensis rats as an appropriate late stage disease-model for trypanosomiasis. Disease progression data showed that survival times were higher in mice infected with KETRI 3439 and KETRI 3622 trypanosomes than in similarly infected rats. However, mice and rats infected with KETRI 3637 had similar high survival times. The host survival time has been estimated between 6 and 9 weeks for EATRO 1989- (Fink & Schmidt, 1979), 3–12 weeks (Kagira et al., 2007) and 3–7 weeks (Kibugu et al., 2009) for KETRI 3741-infected

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T. b. rhodesiense-infected mice. The present host survival data of 4–8 weeks for mice infected with the three trypanosome isolates and rats infected with KETRI 3622 or KETRI 3637 was consistent with the above earlier studies. But the 1–4 weeks survival observed for rats infected with KETRI 3439-infected rats was not. Trypanosome-induced inflammation lesions were more intense in rats. Swiss White mice did not have major pathological changes which is consistent with other reports (Fink and Schmidt 1979). The mice had a more chronic infection (with fewer pathological signs) and are restricted to studies requiring a chronic disease manifestation but not useful for pathological aspects in human sleeping sickness. The KETRI 3637-infected rats had the most severe lesions which normally characterize murine HAT infections. The severe acute infection induced is very consistent with the pathological changes observed in human Rhodesian sleeping sickness. Therefore KETRI 3637-infected M. natalensis rats can be a better model for studying the acute and meningoencephalitic stages of the disease. A further major advantage of Mastomys rats over mice is that the former has a bigger body mass making it possible to monitor changes in the size of organs. Mastomys is also more suitable for blood sample collection for hematological studies (Ojok and Weiss, 1995) which is more problematic in mice due to low circulating blood volume (Kibugu et al., 2010). This is the first report on the susceptibility of Mastomys natalensis rats to T .b. rhodesiense infection. Acknowledgements We thank the Director General, Kenya Agricultural and Livestock Research Organization (KALRO) for granting permission to publish this paper. We are grateful to the following Biotechnology Research Institute staff: Messrs Francis Njunge and John Ndicho, for their technical expertise. This work was funded by Government of Kenya. References Brun, R., Blum, J., Chappuis, F., Burri, C., 2010. Human African trypanosomiasis. Lancet 375, 148–159. Burundi, E.M.E., Karanja, S.M., Njue, A.I., Githiori, J.B., Ndung’u, J.M., 1995. Establishment of a partly DFMO – sensitive primate model of Trypanosoma rhodesiense sleeping sickness. Acta Trop. 59, 71–73. Everitt, B.S., Der, G., 1998. A Handbook of Statistical Analysis Using SAS. Boca Raton, London, New York, Washington D. C. Farah, I.O., Ngotho, M., Kariuki, M., Jeneby, N., Maina, N., Kagira, J.M., Gicheru, M., Han, J., 2014. Handbook of laboratory animal science second edition. In: Hau, J., Steven Schapiro, J. (Eds.), Animal Models for Tropical Parasitic Diseases, Volume IV. CRC Press, Boca raton, Florida. Fink, E., Schmidt, H., 1979. Meningoencephalitis in chronic Trypanosoma brucei rhodesiense infection in the white mouse. Tropenmed. Parasitol. 30, 206–211. Geerts, S., Holmes, P.H., 1998. Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT Technical and Scientific series 1, Food and Agriculture Organization of the United Nations, Rome. Gichuki, C., Brun, R., 1999. Animal models of CNS (second-stage) sleeping sickness. In: Zak, O., Sande, M. (Eds.), Handbook of Animal Models of Infection. Academic Press, London, UK, pp. 795–800. Gutterridge, W.E., Coombs, G.H., 1977. Biochemical mechanisms of drug action. In: Biochemistry of Parasitic Protozoa. The Macmillan Press Ltd., London, Basingstoke, UK, pp. 111–146. Herbert, W.J., Lumsden, W.H.R., 1976. Trypanosoma brucei: a rapid “matching” method for estimating the host’s parasitaemia. Exp. Parasitol. 40, 427–431. Jennings, F.W., Whitelaw, D.D., Urquhart, G.M., 1977. The relationship between duration of infection with Trypanosoma brucei in mice and the efficacy of chemotherapy. Parasitology 75, 143–153. Kagira, J.M., Maina, N., 2007. Occurrence of multiple drug resistance in Trypanosoma brucei rhodesiense isolated from sleeping sickness patients. Onderstepoort J. Vet. Res. 74, 17–22. Kagira, J.M., Ngotho, M., Thuita, J., 2007. Development of a rodent model for late stage Rhodesian sleeping sickness. J. Protozool. Res. 17, 48–56. Kibugu, J.K., Ngeranwa, J.J.N., Makumi, J.N., Gathumbi, J.K., Kagira, J.M., Muchiri, M.W., Mdachi, R.E., 2009. Aggravation of pathogenesis mediated by ochratoxin A in mice infected with Trypanosoma brucei rhodesiense. Parasitology 136, 273–281. Kibugu, J.K., Muchiri, M.W., Mbugua, N., Mwangi, J.N., Thuita, J.K., 2010. Comparative evaluation of anticoagulatory activity of ethylenediamine

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