Veterinary Parasitology 80 (1999) 197±213

Study on the sequential tsetse-transmitted

Trypanosoma congolense, T. brucei brucei and
T. vivax infections to African buffalo, eland,
waterbuck, N'Dama and Boran cattle
S.K. Molooa,*, G.O. Orindab, C.L. Sabwaa,
S.H. Minjaa, R.A. Masakea
a
International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya
b
Kenya Agricultural Research Institute (KARI), National Veterinary Research Centre, Kabete, Nairobi, Kenya
Received 10 February 1998; accepted 14 July 1998

Abstract

Susceptibility of African buffalo, eland, waterbuck, N'Dama and Boran cattle to sequential
Glossina morsitans centralis-transmitted infections of Trypanosoma congolense, T. brucei brucei
and T. vivax was compared, and their possible role as reservoirs of these parasites for G. moristans
centralis, G. pallidipes, G. austeni, G. brevipalpis and G. longipennis determined. The buffalo,
eland, waterbuck and N'Dama controlled T. congolense parasitaemias and were able to prevent
anaemia. By contrast, one Boran became severely anaemic whilst the other controlled parasitaemia
and anaemia. When the above five species of Bovidae were rechallenged with T. brucei brucei they
showed persistent parasitaemias but did not develop anaemia. The buffalo died of other causes.
When the remaining four bovids were rechallenged with T. vivax they became infected with mixed
T. vivax/T. b. brucei parasites. Eland, waterbuck and N'Dama controlled parasitaemias and anaemia
whereas the Boran became anaemic. Cyclical development of T. congolense occurred in
G. moristans centralis when fed on the bovid hosts, with buffalo being infective for tsetse flies
for a much longer period. There was no relationship between the levels of T. congolense
parasitaemia in the bovid hosts and the resultant infection rates in tsetse flies. Glossina m. centralis
was more susceptible than G. pallidipes to T. brucei brucei whilst G. austeni the least;
G. brevipalpis and G. longipennis were refractory to the mature infection. Again there was no
relationship between T. brucei brucei parasitaemia levels in the hosts and infection rates in the flies.
Glossina m. centralis and G. pallidipes showed mixed T. brucei brucei/T. vivax infections whilst
G. austeni, G. brevipalpis and G. longipennis became infected with T. vivax alone. Tsetse flies
showed higher T. vivax infection rates when fed on the hosts with high parasitaemias than those

* Corresponding author. Tel.: +254-2-630743; fax: +254-2-631499; e-mail: d.moloo@cgnet.com

0304-4017/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 2 0 9 - X
198 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

with low parasitaemias. Thus trypanotolerant African buffalo, eland, waterbuck, N'Dama as well as
some trypanosusceptible Boran cattle can serve as reservoirs of single or mixed trypanosome
infections for tsetse flies. This study has also shown that the Ag-ELISA on the sera from the five
bovid hosts had low sensitivity and species-specificity. Examinations of thin wet blood films and
buffy coats with a phase-contrast microscope were not sensitive enough to detect the parasites on all
occasions. Xenodiagnosis using mice for T. brucei brucei and T. congolense infections, and tsetse
flies for all the three trypanosome species were most sensitive for the detection of trypanosome
infections in the bovid hosts. # 1999 Elsevier Science B.V. All rights reserved.

Keywords: Reservoir hosts; Buffalo; Eland; Waterbuck; Cattle-protozoa; Boran; Goat; Mouse; Trypanosoma
congolense; Trypanosoma brucei brucei; Trypanosoma vivax; Glossina morsitans centralis; Glossina pallidipes;
Glossina austeni; Glossina brevipalpis; Glossina longipennis

1. Introduction

African wild mammals have evolved in the environment of tsetse flies and their
associated trypanosomes, and through natural selection over millions of years have
survived and managed to coexist and serve as food source for the flies and reservoir hosts
for the parasites. However, domestic cattle were introduced to Africa as recently as 6000
B.C. (Epstein, 1971) and are therefore susceptible to trypanosomosis, although some
West Africa taurine cattle breeds are relatively resistant to the pathological effects of the
disease due to their duration of exposure to trypanosome infections (Stewart, 1951;
Murray et al., 1982). However, trypanotolerance in such cattle breeds is not of the same
degree as in the African wild Bovidae. Thus, in areas of high trypanosomosis challenge
from tsetse vectors, especially in combination with nutritional stress, trypanotolerant
cattle develop anaemia accompanied by loss of weight and fertility; in extreme cases the
disease results in death of the hosts (Murray and Trail, 1984; Murray et al., 1984).
The African wild Bovidae exhibit a good degree of trypanotolerance (Dwinger et al.,
1986; Grootenhuis et al., 1982, 1990; Moloo et al., 1995), and have been found to carry
natural infections of T. congolense, T. vivax and/or T. brucei parasites (Ashcroft, 1959;
Baker et al., 1967; Geigy et al., 1971) which are causative agents of bovine
trypanosomosis. Moreover, they have a wide geographical distribution in much of sub-
Saharan Africa (Dorst and Dandelot, 1972) where they comprise a significant source of
bloodmeals for many species of tsetse flies (Moloo, 1993) and also serve as important
reservoirs of trypanosome infections for tsetse flies (Moloo and Kutuza, 1974; Moloo et
al., 1971, 1980; Rogers and Boreham, 1973). The pathogenic trypanosome infections in
the wild bovid hosts are spread by tsetse vectors to trypanosusceptible and trypanotolerant
cattle which could in turn also serve as the infection reservoirs for other tsetse flies.
The objective of the present study was to determine the duration, levels of parasitaemia
and packed red cell volume (PCV) profile in African buffalo, eland, waterbuck, N'Dama
and Boran cattle when exposed to the bites of tsetse flies infected with T. congolense, as
well as the susceptibility of Glossina morsitans centralis when fed on these infected hosts
at different time intervals in the course of infection. In addition, these hosts were
sequentially infected with T. brucei brucei and T. vivax using G. moristans centralis and
 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213 199

the parasitaemias, PCV as well as their reservoir potential for these parasites for
G. moristans centralis, G. pallidipes, G. austeni, G. brevipalpis and G. longipennis were
determined.

2. Materials and methods

2.1. Experimental animals

A male African buffalo weighing 375 kg, a female eland (250 kg) and a female
waterbuck (265 kg) were used. The buffalo and waterbuck aged about 4 years, were born
in captivity at the Veterinary Laboratories of the Kenya Agricultural Research Institute
(KARI), Kabete, an area free of tsetse flies and trypanosomosis. A female eland was
captured in Bamburi, Coast Province, Kenya. Thus it was of unknown age and was from
an area known to be infested with tsetse flies and endemic for trypanosomosis. These
animals were kept in a fly-proof isolation building and fed on grass hay supplemented
with lucerne hay and ranch cubes. Mineral salt-licks and drinking water were supplied ad
libitum.
The three-year-old female N'Dama weighing 250 kg and a female Boran (250 kg)
cattle were born at ILRI Kapiti Plains Estate located in Machakos District, Kenya, which
is also free of tsetse flies and trypanosomosis. Throughout the experiment the animals
were housed at the ILRI Campus, Kabete, Kenya, in a fly-proof isolation building and
maintained on water and grass hay with concentrate nutritional supplements.
Adult castrated male goats (East African MaasaiGalla), weighing 20±25 kg, were
reared on the ILRI Campus. The goats were housed and maintained as described for the
cattle.
Outbred adult Swiss white mice, weighing approximately 30 g, were from the ILRI
breeding colony and maintained on an ad libitum supply of a pelleted ration and water.

2.2. Trypanosomes

Trypanosoma congolense stock IL 3947 is a derivative of C-49 which was isolated

from a bovine cow in Trans-Mara District, Kenya, in 1966 (Wellde et al., 1974);
T. congolense clone IL 1180 is a derivative of STIB 212 which was isolated from a lion in
the Serengeti National Park, Tanzania, in 1971 (Geigy and Kauffmann, 1973); and
T. brucei brucei stock IL 375 is a derivative of STIB 247 which was isolated from a
Coke's hartebeest in Serengeti, Tanzania, in 1971 (Geigy and Kauffmann, 1973). This
T. brucei brucei stock is sensitive to human serum (Jenni and Brun, 1982). Trypanosoma
vivax stock IL 3913 is a derivative of GALANA/78/TRYPS 2392 which was isolated
from a Zebu on a farm located between Galana and Malindi, Coast Province, Kenya, in
1978.

2.3. Tsetse flies

Five tsetse species used were from the colonies bred at ILRI. They were G. moristans
centralis originating from the mainland Tanzania; G. pallidipes, G. austeni,
200 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

G. brevipalpis and G. longipennis from Kenya. The rearing techniques for these five
different tsetse species have been described (Moloo et al., 1993). Glossina m. centralis
infected with T. congolense, T. brucei brucei or T. vivax and used to infect the
experimental bovids were produced using the method as described previously (Moloo
et al., 1992).

2.4. Monitoring of parasitaemia and antigenaemia

Blood samples from the experimental bovids were collected weekly from the jugular
vein of each animal into two evacuated tubes, one containing the anticoagulant
dipotassium salt of ethylene diamine tetra-acetic acid (EDTA) for parasite detection and
PCV determination, whereas the other tube was without EDTA for serum preparation.
The degree of parasitaemia was estimated by counting the number of trypanosomes in a
wet thin blood film using a phase-contrast microscope at 400 times magnification
(Herbert and Lumsden, 1976). The PCV of the EDTA blood was determined and the buffy
coat was examined for parasites with a phase-contrast microscope at 400 times
magnification (Murray et al., 1977). Blood was also inoculated into five sub-lethally
irradiated mice at 600 rads, 0.5 ml blood per mouse, and wet thin blood films of the tail
blood were examined for the parasites with a phase-contrast microscope at 400 times
magnification for 40 days. Antigenaemia in serum samples was determined using the
antigen-trapping enzyme-linked immunosorbent assays (Ag-ELISA) as described by
Nantulya and Lindquist (1989).

2.5. Experimental design

Prior to the tsetse challenge, blood was collected from the African buffalo, eland,
waterbuck, N'Dama and Boran cattle, and their PCV, parasitaemia and antigenaemia
were determined. They were found to be aparasitaemic by microscopy and mouse
inoculation but antigenaemic, and therefore they were injected intramuscularly in the
thigh with diminazene aceturate (Berenil1, Hoechst, Germany) at a dose of 10 mg kgÿ1
body weight. Three months after the drug treatment, the five bovids were bled and then
each was infected with T. congolense IL 3947 using 5 G. moristans centralis. After the
detection of infection, the animals were bled regularly at 15- or 30-day intervals for 360
days and on the same days 200 teneral male and 200 teneral female G. moristans
centralis were fed on the flanks of each animal. The tsetse flies were then maintained on
goats and on day 30 after the infected feed, the surviving tsetse were dissected to
determine the trypanosome infection rates.
The Boran became anaemic with PCV of 16% on day 45 post-patent infection and it
was therefore treated with Berenil at a dose of 3.5 mg kgÿ1 body weight. This animal was
replaced by a 3-year old ILRI-bred Boran steer weighing 260 kg, which was bled and
then five G. moristans centralis infected with T. congolense IL 3947 were fed on its flank.
The experiment procedure was then followed as described above.
The waterbuck did not become infected by day 45 and, although it was reinfected with
the same stock using 10 fly bites, the animal still did not show infection. It was reinfected
using five G. moristans centralis infected with T. congolense IL 1180 but the animal
 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213 201

showed transient parasitaemia for only 16 days. A second female waterbuck of similar
age and weighing 270 kg was introduced and five G. moristans centralis infected with
T. congolense IL 3947 were fed on its flank. This animal also did not become infected
and, although the animal was rechallenged with the same T. congolense stock IL 3947
using the bites of 10 G. moristans centralis, it still remained resistant to infection. The
animal was challenged again but with five G. moristans centralis infected with
T. congolense IL 1180. This animal also showed transient parasitaemia for only 15 days.
Teneral G. moristans centralis were allowed to feed on the flanks of this animal during
the period of parasitaemia to determine infectivity of the parasites for G. moristans
centralis.
On day 525 after infection with T. congolense, the African buffalo, eland, waterbuck,
N'Dama and Boran were bled, and each was then rechallenged using the bites of five
G. moristans centralis infected with T. brucei brucei IL 375. The prepatent period in all
the five animals was 6±7 days, and between days 19 and 66 post-infection, teneral male
and female G. moristans centralis, G. pallidipes, G. austeni. G, brevipalpis and
G. longipennis were fed on their shaven flanks. The flies were then maintained on goats
and were dissected on day 30 after the infected feed to determine their typanosome
infection rates. However, the buffalo later strayed into an adjacent pen and was killed by
another buffalo.
On day 649 post-infection with T. congolense, the eland, waterbuck, N'Dama and
Boran were bled, and each was then rechallenged using the bites of 10 G. moristans
centralis infected with T. vivax IL 3913. Parasitaemia was not detected in eland and
waterbuck whilst in the N'Dama and Boran the parasites were detected on days 16 and
13, respectively. Teneral male and female G. moristans centralis, G. pallidipes,
G. austeni, G. brevipalpis and G. longipennis were fed on their flanks between days
19 and 39 post-infection. The flies were maintained on goats and were dissected on day
30 to determine trypanosome infection rates. The four animals were treated with Berenil
at a dose of 7 mg kgÿ1 body weight on day 687 after the T. congolense infection.

3. Results

The prepatent periods of T. congolense infection in the African buffalo, eland, N'Dama
and Boran were 43, 25, 19 and 19 days, respectively. Tables 1±4 show the following.
Trypanosomes were observed by microscopy in the blood of buffalo only on day 15 post-
patent infection (Table 1). Thereafter the buffy coat was negative for the parasites but the
animal was parasitaemic up to day 180 post-infection as revealed by mouse inoculation.
The Ag-ELISA showed circulating T. congolense antigens throughout the experiment, but
the assays also showed circulating T. brucei brucei on 8 and T. vivax antigens on 11 of 14
different days. Its pre-infection PCV was 26%, and thereafter it fluctuated between 25
and 33%.
In the eland the blood was positive by microscopy only on day 60 whilst the animal
was parasitaemic up to day 90 as revealed by mouse inoculation (Table 2). The Ag-
ELISA of the sera from eland revealed circulating T. congolense antigens for all the days
tested, but they also gave positive results for T. brucei brucei on three occasions and
T. vivax antigens on day 360 post-patent infection. Its pre-infection PCV was 41%, it
 202

Table 1
Infection rates in both sexes of Glossina morsitans centralis when fed on an African buffalo infected with Trypanosoma congolense stock IL 3947

Post-patent PCV Parasitaemia Inoculation Antigen-ELISA No. of tsetse Infection rates (%)
(days) (%) in blooda of mice dissected
Tc Tb Tv Midgut Labrum Hypopharynx

15 25 2.5105 5/5 ‡ ÿ ÿ 350 5.1 4.6 4.6

30 29 BCÿ 5/5 ‡ ‡ ‡ 372 6.7 5.6 4.8
45 26 BCÿ 5/5 ‡ ‡ ‡ 375 17.6 14.6 12.5
60 29 BCÿ 5/5 ‡ ‡ ‡ 352 8.2 6.5 5.7
90 32 BCÿ 5/5 ‡ ‡ ‡ 285 10.2 8.8 7.7
120 28 BCÿ 5/5 ‡ ‡ ÿ 330 8.2 7.0 4.8
150 25 BCÿ 4/5 ‡ ÿ ÿ 330 1.5 1.5 1.2
180 23 BCÿ 2/5 ‡ ÿ ‡ 370 3.0 2.2 1.9
210 25 BCÿ 0/5 ‡ ÿ ‡ 342 0.0 0.0 0.0
240 27 BCÿ 0/5 ‡ ‡ ‡ 374 0.3 0.3 0.3
270 30 BCÿ 0/5 ‡ ‡ ‡ 330 0.0 0.0 0.0
300 32 BCÿ 0/5 ‡ ÿ ‡ 351 0.0 0.0 0.0
330 33 BCÿ 0/5 ‡ ‡ ‡ 343 0.0 0.0 0.0
360 32 BCÿ 0/5 ‡ ÿ ‡ 366 0.0 0.0 0.0
a
Parasitaemia in the host are the number of trypanosomes/ml of blood at the time tsetse fed on it.
S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

BCÿ: Infection was not detected in the buffy coat.

Tc: T. congolense, Tb: T. brucei brucei, Tv: T. vivax
Table 2
Infection rates in both sexes of Glossina morsitans centralis when fed on an eland infected with Trypanosoma congolense stock IL 3947

Post-patent PCV Parasitaemia Inoculation Antigen-ELISA No. of tsetse Infection rates (%)
(days) (%) in blooda mice dissected
Tc Tb Tv Midgut Labrum Hypopharynx

15 30 BCÿ 5/5 ‡ ‡ ÿ 366 12.3 11.5 10.4

30 30 BCÿ 5/5 ‡ ÿ ÿ 363 9.4 7.2 6.6
45 32 BCÿ 5/5 ‡ ÿ ÿ 360 20.3 19.4 17.5
60 31 2.5105 5/5 ‡ ÿ ÿ 332 13.0 10.8 9.0
90 35 BCÿ 2/5 ‡ ÿ ÿ 339 5.6 4.1 3.8
120 43 BCÿ 0/5 ‡ ÿ ÿ 364 0.0 0.0 0.0
150 44 BCÿ 0/5 ‡ ÿ ÿ 355 0.0 0.0 0.0
180 43 BCÿ 0/5 ‡ ÿ ÿ 372 0.0 0.0 0.0
210 43 BCÿ 0/5 ‡ ÿ ÿ 368 0.0 0.0 0.0
240 46 BCÿ 0/5 ‡ ‡ ÿ 374 0.0 0.0 0.0
270 42 BCÿ 0/5 ‡ ‡ ÿ 362 0.0 0.0 0.0
300 45 BCÿ 0/5 ‡ ÿ ÿ 373 0.0 0.0 0.0
330 40 BCÿ 0/5 ‡ ÿ ÿ 348 0.0 0.0 0.0
360 46 BCÿ 0/5 ‡ ÿ ‡ 379 0.0 0.0 0.0
S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

a
Parasitaemia in the host is the number of trypanosomes/ml of blood at the time tsetse fed on it.
BCÿ: Infection was not detected in the buffy coat.
203
 204

Table 3
Infection rates in both sexes of Glossina morsitans centralis when fed on a N'Dama infected with Trypanosoma congolense stock IL 3947

Post-patent PCV Parasitaemia Inoculation Antigen-ELISA No. of tsetse Infection rates (%)
(days) (%) in blooda mice dissected
Tc Tb Tv Midgut Labrum Hypopharynx

15 36 5.0105 ND ‡ ‡ ‡ 229 20.1 17.5 16.2

30 30 2.5105 ND ‡ ‡ ‡ 196 14.3 11.2 10.7
45 29 2.5105 ND ‡ ‡ ‡ 232 16.4 10.8 10.3
60 34 BCÿ 0/5 ‡ ‡ ‡ 368 0.0 0.0 0.0
90 38 BCÿ 5/5 ‡ ‡ ÿ 386 1.0 0.5 0.5
120 41 BCÿ 0/5 ÿ ÿ ÿ 337 0.0 0.0 0.0
150 46 BCÿ 0/5 ÿ ÿ ÿ 369 0.0 0.0 0.0
180 43 BCÿ 0/5 ÿ ÿ ÿ 336 0.0 0.0 0.0
210 42 BCÿ 0/5 ‡ ÿ ÿ 378 0.0 0.0 0.0
240 41 BCÿ 0/5 ÿ ÿ ÿ 351 0.0 0.0 0.0
270 46 BCÿ 0/5 ÿ ÿ ÿ 382 0.0 0.0 0.0
300 42 BCÿ 0/5 ‡ ÿ ÿ 384 0.0 0.0 0.0
330 47 BCÿ 0/5 ÿ ÿ ÿ 380 0.0 0.0 0.0
360 47 BCÿ 0/5 ÿ ÿ ÿ 348 0.0 0.0 0.0
a
Parasitaemia in the host is the number of trypanosomes/ml of blood at the time tsetse fed on it.
BCÿ: Infection was not detected in the buffy coat.
S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

ND: Mice inoculation was not done because host animal was parasitaemic by the wet thin blood film examination.
Table 4
Infection rates in both sexes of Glossina morsitans centralis when fed on a Boran steer infected with Trypanosoma congolense stock IL 3947

Post-patent Parasitaemia Inoculation Antigen-ELISA No. of tsetse Infection rates (%)

(days) in blooda mice dissected
Tc Tb Tv Midgut Labrum Hypopharynx

15 2.0106 ND ‡ ‡ ÿ 236 23.7 17.4 15.3

30 2.5105 ND ‡ ‡ ÿ 206 8.7 7.3 6.3
45 2.5105 ND ‡ ‡ ÿ 226 15.0 12.8 11.5
60 2.5105 ND ‡ ‡ ÿ 378 12.2 9.8 6.9
90 BC‡ 5/5 ÿ ÿ ÿ 378 9.5 9.0 9.0
120 BCÿ 1/5 ÿ ÿ ÿ 362 1.4 1.4 1.4
150 BCÿ 0/5 ÿ ‡ ÿ 373 0.0 0.0 0.0
180 BCÿ 0/5 ÿ ÿ ÿ 356 0.0 0.0 0.0
210 BCÿ 0/5 ÿ ‡ ÿ 377 0.0 0.0 0.0
240 BCÿ 0/5 ÿ ÿ ÿ 364 0.0 0.0 0.0
270 BCÿ 0/5 ÿ ÿ ÿ 378 0.0 0.0 0.0
300 BCÿ 0/5 ÿ ÿ ÿ 387 0.0 0.0 0.0
330 BCÿ 0/5 ÿ ÿ ÿ 391 0.0 0.0 0.0
360 BCÿ 0/5 ÿ ÿ ÿ 351 0.0 0.0 0.0
a
Parasitaemia in the host is the number of trypanosomes/ml of blood at the time tsetse fed on it.
S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

BC‡: Infection was detected in the buffy coat.

BCÿ: Infection was not detected in the buffy coat.
ND: Mice inoculation was not done because host animal was parasitaemic by the wet thin blood film examination.
205
206 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

declined to 30% on day 15 and remained around this level up to day 60. Thereafter its
PCV increased and it was 46% on day 360 post-patent infection.
In the N'Dama the blood was positive by microscopy up to day 45 of patent infection,
whilst on day 90 when it was negative by microscopy, its blood infected all the five mice
(Table 3). The Ag-ELISA of the sera from the N'Dama showed circulating T. congolense
antigens for the first 90 days and again on days 210 and 300. The assays also gave
positive results for T. brucei brucei and T. vivax antigens for the first 90 and 60 days,
respectively. Its pre-challenge PCV was 35%, it declined to 29% on day 45 and thereafter
it gradually increased reaching 47% on day 360 post-patent infection.
In the Boran the blood was positive by microscopy up to day 90 post-patent infection
and on day 90 by mouse inoculation (Table 4). On day 120 it was negative by buffy coat
examination but one out of the five mice inoculated with its blood became infected. The
Ag-ELISA assays on the sera of the Boran showed circulating T. congolense antigens up
to day 60, and also T. brucei brucei antigens up to day 60 as well as on days 150 and 210
post-patent infection. Its pre-infection PCV was 33% which declined to 21% on day 45
but thereafter it increased and was 37% on day 360 of infection.
The infection rate data (Tables 1±4) revealed that in the African buffalo T. congolense
stock IL 3947 was infective for G. moristans centralis up to day 240 post-patent
infection, in the eland and N'Dama the parasites were infective for tsetse only up to day
90 whilst in the Boran up to day 120 post-patent infection. There was no relationship
between the parasitaemia levels in the host and T. congolense infection rates in
G. moristans centralis. For example, in the buffalo the parasitaemia was 2.5105 on day
15 post-patent infection and when tsetse were fed on this host on that day the resultant
midgut trypanosome infection rate was 5.1%, whilst on day 45 of infection with
parasitaemia being undetectable by microscopy the infection rate in the midgut of tsetse
flies was higher at 17.6%.
The two waterbucks were completely resistant to infection with T. congolense stock IL
3947 when challenged by five and later by 10 bites of infected G. moristans centralis.
However, when rechallenged by five fly bites of G. moristans centralis infected with
T. congolense clone IL 1180, the two waterbucks showed low and fleeting parasitaemias
for about 15 days but without anaemia. When 400 teneral G. moristans centralis were fed
on one of the waterbucks during patent infection with T. congolense IL 1180, the resultant
infection rates in the midgut, labrum and hypopharynx of the flies were 24.9, 18.4 and
15.8%, respectively.
The prepatent periods of T. brucei brucei infections in the buffalo, eland and waterbuck
were 7 days whilst in the N'Dama and Boran cattle they were 6 days. Their PCV levels
declined by between 8% in the buffalo to 18% in the waterbuck, but they all appeared to
be unaffected by T. brucei brucei infections. The five bovid hosts were parasitaemic with
T. brucei brucei up to day 125 after infection. The Ag-ELISA revealed circulating
T. brucei brucei antigens on seven occasions including in the preinfection serum of the
buffalo, and on three, five, six and two occasions in eland, waterbuck, N'Dama and
Boran, respectively. Also, the assay gave positive results for T. congolense antigens in all
the bovid hosts up to about 600 days after they were infected with T. congolense.
Moreover, the assay also gave results positive for T. vivax antigens in the buffalo and the
N'Dama.
 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213 207

Table 5
Infection rates in five different Glossina species when fed on the five different bovid species infected with
Trypanosoma brucei brucei stock IL 375

Hosts Parasitaemia Tsetse species No. of tsetse Infection rates (%)

in hostsa dissected
Midgut Salivary glands
Buffalo BC‡ G. moristans centralis 370 45.9 8.4
G. pallidipes 345 31.9 3.8
G. austeni 284 7.7 0.0
G. brevipalpis 173 19.1 0.0
G. longipennis 121 24.8 0.0
Eland BC‡ G. moristans centralis 350 51.4 9.7
G. pallidipes 155 30.8 8.4
G. austeni 214 10.3 0.5
G. brevipalpis 106 22.6 0.0
G. longipennis 106 16.0 0.0
Waterbuck 5.0105 G. moristans centralis 399 51.6 11.8
G. pallidipes 327 36.1 9.2
G. austeni 229 5.2 0.0
G. brevipalpis 153 5.9 0.0
G. longipennis 122 13.1 0.0
N'dama 2.5105 G. moristans centralis 342 58.4 13.7
G. pallidipes 396 22.2 3.8
G. austeni 267 13.5 1.1
G. brevipalpis 258 10.9 0.0
G. longipennis 262 17.6 0.0
Boran BC‡ G. moristans centralis 343 59.8 16.0
G. pallidipes 394 19.8 3.6
G. austeni 316 30.1 0.3
G. brevipalpis 216 20.4 0.0
G. longipennis 223 21.1 0.0
a
Parasitaemia in the hosts is the number of trypanosomes/ml of blood at the time tsetse fed on them.
BC‡: Infection detected only in the buffy coat.

Table 5 shows that G. moristans centralis was most susceptible to T. brucei brucei
infection followed by G. pallidipes. Glossina austeni showed very low levels of mature
infections when fed on infected eland, N'Dama or Boran, whilst in both G. brevipalpis
and G. longipennis the infections were arrested in the midguts of the flies. This table also
shows that there was no relationship between the levels of T. brucei brucei parasitaemia
in the bovid hosts and the resultant infection rates in the midguts of different tsetse
species or the same species of tsetse fly. For example, in the waterbuck the T. brucei
brucei parasitaemia was 5.0105 whilst the resultant infection rates in the midgut of five
tsetse species varied between 51.6% in G. moristans centralis and 5.2% in G. austeni.
Also, the higher mature T. brucei brucei infection rates in G. moristans centralis than
G. pallidipes were significant when fed on buffalo (2 ˆ 6.57, p<0.05), the N'Dama
(2ˆ23.6, p<0.01) or the Boran (2ˆ33.67, p<0.01), whilst the differences were not
significant when fed on eland (2ˆ0.22, p>0.09) or waterbuck (2ˆ0.15, p>0.05).
Trypanosoma vivax parasitaemias in the N'Dama and Boran were very high.
Examination of their Giemsa-stained blood slides showed scanty T. brucei brucei
 208

Table 6
Infection rates in five different Glossina species when fed on the four different bovid species infected with Trypanosoma vivax stock IL 3913 and Trypanosoma brucei
brucei stock IL 375

Hosts Parasitaemia Inoculation Tsetse species No. of tsetse Infection rates (%)
in hostsa of mice dissected
Midgut Labrum Hypopharynx Salivary glands
Eland BCÿ 1/5 G. moristans centralis 405 30.6 29.6 24.7 4.4
G. pallidipes 217 15.2 11.5 8.3 0.9
G. austeni 136 0.0 0.0 0.0 0.0
G. brevipalpis 187 14.4 8.0 5.9 0.0
G. longipennis 170 6.5 7.1 4.1 0.0
Waterbuck BCÿ 1/5 G. moristans centralis 410 33.7 35.4 24.4 4.6
G. pallidipes 182 17.6 9.3 8.2 1.6
G. austeni 152 0.0 0.0 0.0 0.0
G. brevipalpis 191 15.2 9.4 8.4 0.0
G. longipennis 152 5.9 8.6 5.3 0.0
2 N'dama 5.0105 ND G. moristans centralis 386 33.4 50.3 39.9 4.7
G. pallidipes 254 24.4 28.0 24.8 1.6
G. austeni 298 0.0 3.0 3.0 0.0
G. brevipalpis 192 29.7 20.8 16.1 0.0
G. longipennis 179 15.1 21.8 16.2 0.0
Boran 3.0107 ND G. moristans centralis 229 0.9 41.9 38.0 0.0
G. pallidipes 135 0.0 77.8 72.6 0.0
G. austeni 166 0.0 15.7 13.9 0.0
S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

G. brevipalpis 108 0.0 81.5 69.4 0.0

G. longipennis 105 0.0 67.6 46.7 0.0
a
Parasitaemia in the hosts is the number of trypanosomes/ml of blood at the time tsetse fed on them.
BCÿ, Infection was not detected in the buffy coat.
ND: Mice inoculation was not done where hosts were parasitaemic.
 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213 209

amongst abundant T. vivax parasites in both the animals. By contrast, the parasitaemias in
the eland and waterbuck were undetectable by microscopy and only one out of five mice
inoculated with the blood of each of these hosts became infected with T. brucei brucei.
The mixed T. brucei brucei/T. vivax infections in eland, waterbuck and N'Dama had no
marked changes in their PCV, whilst the Boran became anaemic with PCV of 18% by day
45 after T. vivax infection. However, in the eland and waterbuck no parasites were
detectable by microscopy and the two hosts showed no circulating T. vivax antigens
although they were parasitaemic since the flies became infected with T. vivax when fed
on them (see Table 6). The Ag-ELISA showed circulating T. congolense antigens in these
two hosts and in the N'Dama throughout this experiment, as well as in the Boran on 3 out
of 7 different days tested, that is, 687 days after they were infected with T. congolense.
Table 6 shows that T. brucei brucei infected the midgut of all the tsetse species except
G. austeni when fed on eland, waterbuck and N'Dama, whilst the parasites infected only
the midgut of two G. moristans centralis when fed on the Boran. Thus, T. brucei brucei
parasites were still present in circulation of all the four bovid hosts 150 days after they
were infected with this parasite. This table also shows that G. moristans centralis and
G. pallidipes carried mixed mature T. vivax/T. brucei brucei infections, at a higher rate in
the former tsetse, when fed on the eland, waterbuck and N'Dama. The T. vivax infection
rates were generally higher in all the five tsetse species when fed on the N'Dama or
Boran in which T. vivax parasitaemias were very high as compared to the flies which were
fed on the eland and waterbuck in which T. vivax parasitaemias were undetectable by
microscopy.

4. Discussion

The African buffalo, eland and waterbuck did not show clinical signs of
trypanosomosis following sequential infection with T. congolense, T. brucei brucei and
T. vivax using the bites of infected G. moristans centralis. The buffalo and eland were
parasitaemic with T. congolense stock IL 3947 for about 240 and 90 days, respectively,
whilst the two waterbucks exhibited complete resistance to these parasites, but upon
rechallenge with T. congolense clone IL 1180 they showed a low level of transient
parasitaemias. The buffalo and eland controlled the parasitaemias and, furthermore, all
the three wild bovid hosts showed delayed prepatent periods with no signs of anaemia.
The trypanotolerant N'Dama showed infection characteristics similar to those of the
infected eland. The study also showed that in one Boran, T. congolense parasitaemia was
high with severe anaemia and therefore the animal had to be treated in order to prevent
death, whilst in the other Boran the red blood cell level declined by 36% in the first 45
days of infection, but thereafter the animal controlled the parasitaemia and at the same
time recovered from mild anaemia. Thus, whereas one Boran was highly susceptible to
congolense-trypanosomosis the other exhibited trypanotorelant traits similar to the
N'Dama. In a previous study (Murray et al., 1981), when 10 N'Dama and 10 Zebu cows
were exposed to a natural field challenge from G. moristans submorsitans, all the Zebu
died whilst only three N'Dama died of trypanosomiasis within 8 months of first exposure.
There is also within-breed variation in susceptibility to T. congolense infection in Boran
and N'Dama cattle (Paling et al., 1991).
210 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213

The study also showed that when the five bovid hosts were rechallenged with T. brucei
brucei using the bites of infected G. moristans centralis, all the animals became infected
with similar prepatent periods, persistent parasitaemias and declining red blood cell levels
in the course of infections, but no signs of anaemia. It is noteworthy that the sera of eland,
waterbuck (Rickman, 1981) and African buffalo (Reduth et al., 1994) were found to have
a trypanocidal factor against T. brucei brucei, yet the results here showed that T. brucei
brucei parasitaemias were persistent in these bovid hosts.
When the four bovid hosts were later rechallenged with tsetse-transmitted T. vivax they
became infected with this parasite together with the previous T. brucei brucei infections.
The mixed T. vivax/T. brucei brucei infections did not appear to affect eland and
waterbuck as their parasitaemias remained very low and they showed no signs of
anaemia. By contrast, the N'Dama and Boran showed high T. vivax parasitaemias.
Whereas the N'Dama was able to prevent anaemia the Boran became affected by the
mixed vivax/brucei-trypanosomosis with severe anaemia and had to be treated in order to
prevent its possible death. Such mixed trypanosome infections have been encountered in
the field in wild bovids (Ashcroft, 1959; Baker et al., 1967; Geigy et al., 1971) as well as
in cattle (Mwambu and Mayende, 1973; Nyeko et al., 1990).
Cyclical development occurred in G. moristans centralis when fed on trypanotolerant
African buffaloes, eland, N'Dama or the trypanosusceptible Boran cattle infected with
T. congolense stock IL 3947, with buffalo being infective for tsetse for a longer period
compared to the other four bovid hosts. There was no relationship between the levels
of parasitaemia in the bovid hosts and the resultant infection rates in the midgut of
G. moristans centralis. Whereas the two waterbucks did not become infected with
T. congolense IL 3947, they showed low and fleeting parasitaemias with T. congolense IL
1180 and one of the two waterbucks was infective for G. moristans centralis when fed
on it. The results also show that cyclical development of T. brucei brucei occurred in
G. moristans centralis, G. pallidipes and in a few G. austeni when they were fed on
the infected bovid hosts. Glossina moristans centralis was more susceptible than
G. pallidipes to T. brucei brucei, whilst G. austeni the least. Glossina brevipalpis and
G. longipennis were not susceptible to the mature T. brucei brucei infection. Again, there
was no relationship between the parasitaemia levels in the hosts and the infection rates in
the midgut of tsetse flies.
When the five tsetse species were fed on eland, waterbuck, N'Dama or Boran cattle
infected with the mixed T. vivax/T. brucei brucei infections, only G. moristans centralis
and G. pallidipes showed mixed mature trypanosome infections. Glossina austeni was
less susceptible to T. brucei brucei infection whereas the two fusca group tsetse were
refractory to mature infection of this parasite. Thus tsetse species which are susceptible to
both T. brucei brucei and T. vivax, such as G. moristans centralis, are also susceptible to
such mixed infections whereas G. brevipalpis and G. longipennis which are susceptible to
T. vivax but refractory to T. brucei brucei infections are not capable of carrying such
double infections. Mixed trypanosome infections have been encountered in the field in
G. moristans submorsitans, G. tachinoides (Llyod et al., 1924) and G. pallidipes (Majiwa
and Otieno, 1990) and it has been shown that tsetse carrying mixed trypanosome
infections can transmit such infections to susceptible hosts (Moloo et al., 1982). The five
tsetse species showed variable rates of mature T. vivax infections and, contrary to
 S.K. Moloo et al. / Veterinary Parasitology 80 (1999) 197±213 211

T. congolense and T. brucei brucei, the infection rates by T. vivax were generally higher in
flies fed on the hosts with higher T. vivax parasitaemias, such as N'Dama or Boran cattle,
than those fed on the hosts with undetectable T. vivax parasitaemias as in eland and
waterbuck.
The results herein show that neither the wild Bovidae nor the N'Dama succumbed to
the sequential infections of T. congolense, T. brucei brucei and T. vivax, and that
trypanotolerant African buffalo, eland, waterbuck and N'Dama as well as some
trypanosusceptible Boran cattle can serve as reservoirs of single or mixed trypanosome
infections for Glossina species. In the field, tsetse flies become infected with a single or
mixed trypanosome infection after feeding on the infected wild mammals, and a fly can
pick up mixed infections by feeding on one or on different infected hosts. When cattle are
allowed to encroach upon the tsetse-infested areas where trypanosomosis is endemic, they
become exposed to the disease. The level of risk of trypanosomosis to cattle will depend
on the frequency with which tsetse flies feed on them, that is, the degree of fly±cattle
contact. In areas of high game density tsetse flies normally feed mostly on them whereas
in areas of low density natural hosts, tsetse flies attack cattle more frequently resulting in
high fly±cattle contact and thus high disease endemicity. Infected cattle can also serve as
reservoirs for trypanosomes and the infection can spread within the cattle herd by
infected tsetse flies.
The work described here has also shown that the monoclonal antibody-based antigen-
detection enzyme immunoassay (Ag-ELISA) when used for the detection of
T. congolense, T. brucei brucei or T. vivax/T. brucei brucei antigens in the sera of
experimentally infected Bovidae had low sensitivity and species-specificity. The
examination of thin wet blood films as well as buffy coat was also unsatisfactory as
they were not sensitive enough to detect trypanosome infections in the infected bovid
hosts on many occasions. However, xenodiagnosis using mice or tsetse flies was quite
sensitive for the detection of trypanosome species infections, but these methods are not
suitable for field application as they are cumbersome and expensive, and moreover
T. vivax and some isolates of T. congolense do not infect mice. Therefore, there is still
need for simple, sensitive and species-specific tests for the diagnosis of animal
trypanosomiasis in livestock in the field.

Acknowledgements

We thank Professor Max Murray of the University of Glasgow, Dr. A.D. Irvin, Dr.
Subhash Morzaria and Dr. A.J. Musoke of ILRI for helpful comments on the manuscript.
We also thank Miss Joyce Maina for typing the manuscript. This is ILRI publication
number 97064.

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