Transboundary and Emerging Diseases

ORIGINAL ARTICLE

Distribution and Application of ELISA for the

Seroprevalence of Tick-Borne Diseases in Central Equatoria
State, Sudan
D. A. Salih1, S. M. Hassan2, I. I. Julla3, M. N. Kyule4, K-H. Zessin4 and A. M. El Hussein1
1
Central Veterinary Research Laboratories, Al Amarat, PO Box 8067, Khartoum, Sudan
2
Department of Parasitology, Faculty of Veterinary Medicine, University of Khartoum, PO Box 32, Khartoum North, Sudan
3
College of Veterinary Science, University of Bahr El Ghazal, PO Box 10739, Khartoum, Sudan
4
Faculty of Veterinary Medicine, University of Berlin, Königsweg 67, D-14163 Berlin, Germany

Keywords:
Anaplasma marginale; Babesia bigemina;
ELISA; seroprevalence; Southern Sudan;
Theileria parva; Theileria mutans
Correspondence:
D. A. Salih. Central Veterinary Research
Laboratories, Al Amarat, PO Box 8067,
Khartoum, Sudan. Tel.: +249 183 380015;
Fax: +249 183 380011;
E-mail: diaeldin2000@hotmail.com
Received for publication February 6, 2008
doi:10.1111/j.1865-1682.2008.01032.x
Summary
This study was carried out to estimate the prevalence of Theileria parva, Theile-
ria mutans, Babesia bigemina and Anaplasma marginale antibodies among natu-
rally infected cattle in Central Equatoria State, Southern Sudan using indirect
enzyme-linked immunosorbent assay (ELISA). Three locations (Khor Rumla,
Nyaing and Gumbo) were selected and surveyed every 3 months during the
period from January to December 2005. Six cattle herds in the three locations
were visited four times during the study period. During each visit, 150 serum
samples were collected from indigenous cattle of both sex and of different age
groups. Assessment of antibodies against tick-borne diseases was carried out
using indirect ELISA. The overall seroprevalence rates of T. parva, T. mutans,
B. bigemina and A. marginale were found to be 58.2%, 88.9%, 51.1% and
37.8% respectively. The highest seroprevalence of T. parva was observed in
Gumbo (69%). An overall seroprevalence of 43.3% for T. parva was reported
in July which was significantly lower than other seasons. Older animals showed
higher seroprevalence than younger ones. The implications of these results on
the epidemiology of tick-borne diseases are discussed with emphasis on East
Coast fever.

Anaplasma marginale, the main tick vectors of which are

Introduction
Tick-borne diseases (TBDs) are the most prevalent and
exert the greatest impact on livestock in tropical and
subtropical regions (Jongejan and Uilenberg, 2004). In
the Sudan, TBDs are considered to be a major con-
straint to dairy development and cause substantial eco-
nomic losses (Gamal and El Hussein, 2003). In Southern
Sudan, the most important TBD is East Coast fever
(ECF) (Theileria parva infection of cattle) transmitted
transstadially by Rhipicephalus appendiculatus, a three-
host tick that infests 1.4% of the total area in the Sudan
(Lessard et al., 1990). The economic losses due to ECF
have been estimated in 11 African countries for the year
1989 to be US$168 million (Mukhebi et al., 1992).
Other TBDs of lesser importance in cattle in Southern
Sudan are Theileria mutans, Babesia bigemina, and
Amblyomma variegatum, Boophilus decoloratus and
Boophilus annulatus respectively (Anonymous, 1983).
However, knowledge about TBDs is still fragmentary in
Southern Sudan.
Thus far, epidemiological studies on TBDs conducted
in Southern Sudan were based on indirect fluorescent
assay (IFA) (Morzaria et al., 1981; Julla, 1994), high-
lighting the need for application of more specific, sensi-
tive, reproducible and easy to perform laboratory
techniques, such as enzyme-linked immunosorbent assay
(ELISA). Recently, reverse line blot (RLB) was applied
to study the epidemiology of TBDs in the region (Salih
et al., 2007). Nonetheless, assessment of antibodies
against TBDs is an important parameter to assess the
endemic situation of these diseases in the region (Perry
and Young, 1995).

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Journal compilation ª 2008 Blackwell Verlag • Transboundary and Emerging Diseases. 55 (2008) 257–262 257
Application of ELISA
The present study was initiated with the aim to esti-
mate the prevalence of serum antibodies to TBDs using
indirect ELISA among cattle in Central Equatoria State,
Southern Sudan. A longitudinal study design was used to
address the question of whether or not endemic stability
to ECF exists in the region.
Materials and Methods
Study area
The study was conducted over 12 months from January
to December 2005 around Juba (4º50¢N, 31º35¢E), the
capital of Central Equatoria State, Southern Sudan. The
ecological zone includes savannah woodland, green belt
and thick forests. The climate of the study area is semi-
equatorial, with minimal variations in seasonal tempera-
tures. The highest mean maximum ambient temperature
occurs during January in dry season with daily maximum
temperatures ranging between 30C and 32C. The mean
minimum occurs during the rainy season, March–
November, with temperatures ranging between 19C
and 27C and rainfall between 900 and 1200 mm for
6–9 months, which usually commences in March
(Julla, 1994).
Study design
A longitudinal survey, with a visit every 3 months, to
three locations was conducted. The locations surveyed
were Khor Rumla (15 km south of Juba), Nyaing (10 km
north of Juba) and Gumbo (10 km east of Juba). Three
cattle herds in Khor Rumla, two in Nyaing and one in
Gumbo, were regularly investigated at each visit. Selection
of herds was made randomly and the formal mechanism
used was lottery. The visits were conducted in January
(dry season), April (after the rain commenced), July
(peak of rainy season) and October (towards the end of
rainy season). From herds 1 and 3, 20 cattle/herd were
sampled, from herds 4 and 5, 30 cattle/herd and from
herds 2 and 6, 25 cattle/herd were sampled. In total, dur-
ing each visit 150 sera were collected. Four age groups of
cattle were sampled viz: <1, 1–2, 2–3 and >4 years old.
All animals sampled were local long-horned nilotic type
Zebu cattle. As a herd-based study and not a survival
study, the same herd was visited and the same number of
samples were collected, but not from the same animals.
Blood was collected in vacutainers, stored in an ice box
and transferred to Juba regional veterinary laboratory
within 4 h after collection. The next day, serum was sepa-
rated by centrifugation. Each serum sample was collected
using a sterile Pasteur pipette into an eppendorf tube,
labelled indicating location, date and animal number, and
then stored at )20C until use.
258 Journal compilation ª 2008 Blackwell Verlag • Transboundary and Emerging Diseases. 55 (2008) 257–262
D. A. Salih et al.
ELISA conditions
Assessment of antibodies against T. parva, T. mutans,
B. bigemina and A. marginale was conducted using ELISA
kits (Svanova, Uppsala, Sweden) according to the
instructions in the manual. For T. parva, the polymorphic
immunodominant molecule (PIM) recombinant antigen
was used (Katende et al., 1998). The optical density (OD)
was used to compute the per cent positivity (PP), using
the formula mean OD (sample or negative control)
divided by the mean OD value of positive control multi-
plied by 100. Per cent positivity of 15% or above was
considered positive (Katende et al., 1998). All samples
were subjected to this ELISA procedure.
For assessment of antibodies against T. mutans, B. bige-
mina and A. marginale, ELISAs described by Katende
et al. (1990), Tebele et al. (2000) and Morzaria et al.
(1999), respectively, were used. For T. mutans, the 32-kD
protein ELISA was used, whereas for B. bigemina, the
200-kD antigen ELISA was used, and for A. marginale,
the 19-kD protein ELISA was used. In the case of
T. mutans, PP of 20% or above was considered positive,
whereas PP of 40% or above was considered positive for
B. bigemina and PP of 25% or above for A. marginale.
For logistical reasons, only 90 samples from the first
visit (January) collected from Khor Rumla (65 samples)
and Gumbo (25 samples), were subjected to T. mutans,
B. bigemina and A. marginale ELISAs.
Adjustment for test misclassification
The Rogan and Gladen (1978) estimator (PRG) was used
to correct the apparent prevalence to true prevalence,
PRG = (AP + Sp ) 1)/(Se + Sp ) 1), where AP is appar-
ent prevalence; Sp, specificity; Se, sensitivity. PRG is left
(0.0) and right (1.0) censored for values lesser than 0 and
greater than 1 respectively.
The sensitivity and specificity of the PIM ELISA for
T. parva were reported to be 99% and 98% respectively
(Katende et al., 1998), whereas sensitivity and specificity
for the 32-kD protein ELISA test for T. mutans have been
estimated to be 99%, while for the B. bigemina, 200-kD
antigen ELISA sensitivity of 97% and specificity of 98%
were reported. The sensitivity and specificity of the 19-kD
protein ELISA for A. marginale have been estimated to be
90% (Morzaria et al., 1999).
Statistical analysis
The univariate chi-squared test was used to test the null
hypothesis that there were no significant differences
between the seroprevalence in different locations, herds,
age groups and seasons. P-values < 0.05 were considered
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D. A. Salih et al. Application of ELISA

significant. Chi-squared (v2) values and degrees of free-
dom (df) are also presented.
Results
Seroprevalence of T. parva
The overall seroprevalence of T. parva was found to be
58.2% (Table 1). After adjustment for test misclassifica-
tion, a true prevalence of 57.9% was reported. Gumbo
(69%) reported the highest seroprevalence followed
by Khor Rumla (63.1%) and Nyaing (48.3%), with the
latter being significantly lower (v2 = 16.936, df = 2,
P < 0.001). Table 2 shows that the seroprevalence
reported among age group 2–3 years old (68.1%) was
the highest, which was not significantly different from
age group >4 years old, but significantly different from
other age groups (v2 = 12.204, df = 3, P < 0.05). In
Table 3, the distribution of T. parva in different age
groups and seasons is presented. The most affected age
group by the season of collection is age group 1–2 years
old, where in July, a reported seroprevalence of 29.3%
among this age group was the lowest seroprevalence
among all age groups in all seasons. An overall seropre-
valence of 43.3% was reported in July which was signifi-
cantly lower than in other seasons (v2 = 21.939, df = 3,
P < 0.001).
Seroprevalence of T. mutans
The overall seroprevalence of T. mutans was found to be
88.9% (Table 1). After adjustment for test misclassifica-
tion, a true prevalence of 89.7% was reported. The high-
est seroprevalence (93.8%) of T. mutans was observed in
Khor Rumla (v2 = 5.82, df = 1, P < 0.05). There was no
significant difference between all age groups tested
(v2 = 3.60, df = 3, P > 0.05).

Table 1. Prevalence of Theileria parva, Theileria mutans, Babesia bigemina and Anaplasma marginale antibodies using indirect ELISA among cattle
in three locations in Central Equatoria State, Southern Sudan during the year 2005

Locations Theileria parva (P/E) Theileria mutans (P/E) Babesia bigemina (P/E) Anaplasma marginale (P/E)

Khor Rumla 63.1% (164/260) 93.8% (61/65) 50.8% (33/65) 33.8% (22/65)
Nyaing 48.3% (116/240) ND ND ND
Gumbo 69% (69/100) 76% (19/25) 52% (13/25) 48% (12/25)
Total 58.2% (349/600) 88.9% (80/90) 51.1% (46/90) 37.8% (34/90)

P/E, number positive/number examined; ND, not done.

Table 2. Comparison of Theileria parva, Theileria mutans, Babesia bigemina and Anaplasma marginale antibodies using indirect ELISA in four age
groups of cattle in Central Equatoria State, Southern Sudan during the year 2005

Age groups(years) Theileria parva (P/E) Theileria mutans (P/E) Babesia bigemina (P/E) Anaplasma marginale (P/E)

<1 50.2% (103/205) 81.6% (31/38) 52.6% (20/38) 28.9% (11/38)

1–2 56.4% (92/163) 95.2% (20/21) 66.7% (14/21) 38.1% (8/21)
2–3 68.1% (81/119) 93.3% (14/15) 40% (6/15) 53.3% (8/15)
>4 64.6% (73/113) 93.8% (15/16) 37.5% (6/16) 43.8% (7/16)
Total 58.2% (349/600) 88.9% (80/90) 51.1% (46/90) 37.8% (34/90)

P/E, number positive/number examined.

Table 3. Distribution of Theileria parva antibodies using indirect ELISA among four age groups of cattle during four different seasons in Southern
Sudan in the year 2005

Seasons
Age groups
(years) January (P/E) April (P/E) July (P/E) October (P/E) Total (P/E)

<1 57.6% (38/66) 52.2% (24/46) 42.3% (22/52) 46.3% (19/41) 50.2% (103/205)
1–2 75.8% (25/33) 58.7% (27/46) 29.3% (12/41) 65.1% (28/43) 56.4% (92/163)
2–3 68% (17/25) 80.6% (25/31) 55.6% (15/27) 66.7% (24/36) 68.1% (81/119)
>4 73.1% (19/26) 88.9% (24/27) 53.3% (16/30) 46.7% (14/30) 64.4% (73/113)
Total 66% (99/150) 66.7% (100/150) 43.3% (65/150) 56.7% (85/150) 58.2% (349/600)

P/E, number positive/number examined.

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Journal compilation ª 2008 Blackwell Verlag • Transboundary and Emerging Diseases. 55 (2008) 257–262 259
Application of ELISA D. A. Salih et al.

T. parva and B. bigemina, and two for both T. parva and

Seroprevalence of B. bigemina
A. marginale antibodies. Antibodies against T. parva alone
The overall seroprevalence of B. bigemina was found to were detected in four animals, five animals were found
be 51.1% (Table 1). After adjustment for test misclassifi- positive for T. mutans alone and one for B. bigemina
cation, a true prevalence of 51.7% was reported. The alone, while no animal was found positive for A. margin-
highest seroprevalence (52%) was reported in Gumbo, ale alone. One animal was found negative for all TBDs
but it was not significantly different from Khor Rumla examined (Table 4).
(v2 = 0.01, df = 1, P > 0.05). There was no significant
difference between all age groups tested (v2 = 4.0, df = 3,
Discussion
P > 0.05).
The justification for this study was the paucity in epide-
miological studies in Southern Sudan, where only two
Seroprevalence of A. marginale
investigations have been carried out on the seropreva-
The overall seroprevalence of A. marginale was found to lence of TBDs (Morzaria et al., 1981; Julla, 1994). In
be 37.8% (Table 1). After adjustment for test misclassifi- the former study, a seroprevalence of 25.8%, 81.9%
cation, a true prevalence of 34.1% was reported. The and 62.7% was reported for T. parva, T. mutans and
highest seroprevalence (48%) was reported in Gumbo, B. bigemina respectively (Morzaria et al., 1981). In the
which was not significantly different from Khor Rumla, latter study, a seroprevalence of 44.2%, 35%, 76.7% and
(v2 = 1.0, df = 1, P > 0.05). There was no significant dif- 84% was reported for T. parva, T. mutans, B. bigemina
ference between all age groups tested (v2 = 3.05, df = 3, and A. marginale respectively (Julla, 1994). In the present
P > 0.05). study, seroprevalence of 58.2%, 88.9%, 51.1% and 37.8%
was reported for T. parva, T. mutans, B. bigemina and
A. marginale respectively. These differences may illustrate
Mixed infections
the performance of different serological tests used in
The cross tabulation of 90 samples tested for all four each study, as in the previous studies, the indirect
TBDs, revealed nine different combinations of mixed fluorescence antibody (IFA) test was used and in the
infections (Table 4). Sixteen animals were positive for the current study recombinant protein-based indirect ELISA
four parasites, 22 animals had antibodies against T. parva was performed. Moreover, the previous studies used a
and T. mutans, while two animals were positive for both cross-sectional study design, whereas a longitudinal study
design was applied here. Also there are differences in
study area, as in the previous studies, the samples were
Table 4. Summary of results obtained for Theileria parva, Theileria collected from Equatoria region, whereas in this study
mutans, Babesia bigemina and Anaplasma marginale antibodies using sampling was restricted to Central Equatoria State, partic-
indirect ELISA among 90 cattle in Central Equatoria State, Southern ularly around Juba town. Setting aside the inherent
Sudan during January 2005
differences in the tests used, the main change to the
Tick-borne diseases No. seroprevalence previous studies was found with respect to T. parva anti-
bodies, which increased from 25.8% in 1981 to 44.2% in
Theileria parva 4
1994, while in 2005 they were 58.2%. This finding may
Theileria mutans 5
Babesia bigemina 1
indicate that ECF has become well established in Central
Theileria parva and Theileria mutans 22 Equatoria State, a fact which is supported by the recent
Theileria parva and Babesia bigemina 2 finding that the main tick vector, Rhipicephalus appendi-
Theileria parva and Anaplasma marginale 2 culatus, has now firmly established in the region (Salih
Theileria mutans and Babesia bigemina 6 et al., 2008). Recently, RLB for detection and differentia-
Theileria mutans and Anaplasma marginale 1 tion of all known Theileria and Babesia species, was
Theileria parva, Theileria mutans and 15
applied to study the epidemiology of TBDs in the region
Babesia bigemina
Theileria parva, Theileria mutans and 11
(Salih et al., 2007). In comparison to the ELISA results, it
Anaplasma marginale was found that RLB reported a much higher infection
Theileria mutans, Babesia bigemina and 4 with T. parva, with 71.2% (427/600) of the animals being
Anaplasma marginale reported as positive compared to 58.2% of the same ani-
Theileria parva, Theileria mutans, Babesia 16 mals reported positive by ELISA. The two tests did report
bigemina and Anaplasma marginale a similar result in January (dry season), but during all
Negative 1
other seasons, the level of antibodies was less that of the
Total 90
parasite DNA as detected by RLB.

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260 Journal compilation ª 2008 Blackwell Verlag • Transboundary and Emerging Diseases. 55 (2008) 257–262
D. A. Salih et al.
The term ‘endemic stability’ has been defined by
Norval et al. (1992) as a ‘climax relationship between
host, vector and environment in which all coexist with
the virtual absence of clinical disease while endemic insta-
bility means incomplete relationship (between host, vector
and environment) in which clinical disease occurs’. Ende-
mic stability for ECF is more likely to exist where the
prevalence of serum antibodies to T. parva is high
(‡70%), whereas an unstable state is characterized by low
(<30%) antibody prevalence (Norval et al., 1992; Perry
and Young, 1995). Taking together the results of seropre-
valence for T. parva reported in this study and the result
of vector distribution in the same area during the same
period (Salih et al., 2008), it may be concluded that an
unstable endemic state for ECF probably exists in the
region. The overall seroprevalence of T. parva was
reported to be 58.2%, which implies that the pool of sus-
ceptible animals may be sufficient for an outbreak of ECF
to occur. An interesting observation was the lowest sero-
prevalence of T. parva in July, a finding suggesting that
an outbreak may probably occur during this month. On
the other hand, if the seroprevalence reported in each
location is taken into account separately, it may suggest
that an endemic stable state exists in Khor Rumla and
Gumbo but not in Nyaing. For T. mutans, an overall
seroprevalence of 88.9% was reported, which is suggestive
of endemic stability. On the other hand, an unstable
endemic state may exist for B. bigemina and A. marginale
in the region depending on the low seroprevalence
reported herein.
Older animals were revealed to have higher prevalence
of T. parva antibodies than the younger ones. This may
be attributed to the fact that the continuous challenge of
animals produced more antibodies, and that the level of
antibodies increased with age (Swai et al., 2005). As there
was only one breed of cattle sampled during this study,
no relationship between TBDs seroprevalence and breed
could be assessed. However, recent work by Ndungu et al.
(2005) on the susceptibility of different cattle breeds to
T. parva infection indicated that zebu cattle were highly
successful in developing a higher degree of resistance than
either Boran or Friesian cattle. This point should be taken
into account when foreign breeds of cattle are introduced
to meet the increasing demand for milk as a result of
re-settling of displaced people after the end of the civil
war.
The high level of seroprevalence of T. mutans detected
during this study raises the question about the specificity of
the 32-kD protein ELISA. Owing to the statement made by
Sugimoto and Fujisaki (2002) that, in endemic areas, all
calves acquire T. mutans infection early in life and remain
life-long carriers, this doubt could be ruled out. Also, A.
variegatum, the main field vector of T. mutans, was
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Journal compilation ª 2008 Blackwell Verlag • Transboundary and Emerging Diseases. 55 (2008) 257–262
Application of ELISA
reported as the predominant tick species in the region all
round the year (Salih et al., 2008), which may support the
observation of high seroprevalence of T. mutans.
The apparent seroprevalence is affected by the sensitiv-
ity and the specificity of the test used (Greiner and Gard-
ner, 2000). Adjustment for test misclassification and
estimation of seroprevalence revealed that the PIM ELISA
for detection of T. parva antibodies and the 19-kD ELISA
for A. marginale overestimated the true prevalence, while
the 32-kD ELISA for T. mutans and 200-kD ELISA for
B. bigemina underestimated the true prevalence. As no
perfect test with 100% sensitivity and specificity exists,
adjustment for test misclassification should be considered.
From the results of the present study, it is concluded
that a high proportion of cattle in Southern Sudan have
been exposed to TBDs suggesting that preventive mea-
sures such as tick control practices undertaken in the
region were ineffective. This conclusion may justify the
need for control methods for TBDs to be implemented in
the region, such as infection and treatment method.
However, it is of great importance to assess the economic
impact of TBDs in the region, before such control meth-
ods are to be applied.
Acknowledgements
The participation of the farmers in Central Equatoria
State is highly appreciated. The professional assistance of
Dr Wani Lako and Mr John Wani in the field samples
collection is gratefully acknowledged. We thank Ulrike
Seitzer and Jabbar Ahmed, Research Center Borstel, for
critically reading the manuscript. This research was sup-
ported by the International Foundation for Science,
Stockholm, Sweden and Organisation of Islamic Confer-
ence Standing Committee on Scientific and Technological
Cooperation (COMSTECH), Islamabad, Pakistan,
through a grant (IFS grant 3765-1) to Mr Diaeldin
Ahmed SALIH. This work is published with the kind per-
mission of the Director-General, Animal Resources
Research Corporation, Sudan. Supported in part by
EU-FP6 INCO-DEV INCOME (INCO-CT-2005-515915).
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ª 2008 The Authors