Transboundary and Emerging Diseases

SHORT COMMUNICATION

Comparison Between Reverse Line Blot and Enzyme-linked

Immunosorbent Assay in Diagnosis of Major Tick-Borne
Diseases of Cattle in Southern Sudan
D. A. Salih1,2, A. R. M. EL Hussein2, J. Ahmed1 and U. Seitzer1
1
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Borstel, Germany
2
Central Veterinary Research Laboratories, Al Amarat, Khartoum, Sudan

Keywords:
enzyme-linked immunosorbent assay; reverse
line blot; Southern Sudan; tick-borne diseases
Correspondence:
U. Seitzer, Division of Veterinary Infection
Biology and Immunology, Research Center
Borstel, Parkallee 22, D-23845 Borstel,
Germany. Tel.: +49 (0) 4537 188413;
Fax: +49 (0) 4537 1886273;
E-mail: useitzer@fz-borstel.de
Received for publication July 3, 2009
doi:10.1111/j.1865-1682.2010.01103.x
Summary
The performance of reverse line blot (RLB) in detecting DNA of Theileria
parva, Theileria mutans and Babesia bigemina was assessed in comparison with
specific antibody detection using indirect enzyme-linked immunosorbent assays
(ELISA) for the same parasites. Among 90 field samples from Central Equatoria
state, Southern Sudan, ELISA reported more positive samples than RLB did.
The concordance of RLB showed 66.7%, 81.1% and 48.9% relative to the
results of ELISA for T. parva, T. mutans and B. bigemina respectively. It has to
be borne in mind that the results of ELISA might represent previous infections,
while that of RLB would not only reflect an active infection, but also a carrier
status. Therefore, the selection of the test would depend on the specific aims of
the study.

sensitivity and specificity can be achieved by combining

Introduction
Tick-borne diseases (TBD) represent a major obstacle to
livestock development in Southern Sudan. Major TBD of
cattle in Southern Sudan are Theileria parva, Theileria
mutans and Babesia bigemina (El Hussein et al., 2004). In
Southern Sudan, Rhipicephalus appendiculatus was
reported to be the most efficient vector of T. parva, while
Amblyomma lepidum was reported to be the vector of
T. mutans and Boophilus spp. as the vector for Babesia
infection (Anonymous 1983).
The most commonly used serum-antibody assay for
assessment of TBD antibodies has been the indirect
fluorescent-antibody assay (IFA) (Burridge, 1971); how-
ever, it is tedious and subjective, and cross-reactions are
considered a problem (Kiltz et al., 1986). Enzyme-linked
immunosorbent assays (ELISA) have the advantage over
IFA of being less laborious and many samples can be
tested quickly. Several polymerase chain reaction (PCR)-
based diagnostic procedures for detection of TBD have
been developed (Bishop et al., 1992). However, increased
PCR with a specific hybridization by means of reverse line
blot (RLB), a macroarray that is also capable of identifying
mixed infections from one sample (Gubbels et al., 1999).
The current study aimed at comparing the performance
of RLB and ELISA in the diagnosis of T. parva, T. mutans
and B. bigemina of cattle in Southern Sudan.
Materials and Methods
A total of 90 samples (blood and serum) were collected
from cattle in Central Equatoria State, Southern Sudan
during January 2005. Samples were collected from female
in one herd in Khor Rumla area (15 km south from Juba,
the capital of Central Equatoria State). Samples were pro-
cessed for molecular detection of T. parva, T. mutans and
B. bigemina using RLB (Salih et al., 2007) as well as anti-
bodies assessment using specific indirect ELISA (Salih
et al., 2008). For T. parva the polymorphic immunodomi-
nant molecule recombinant antigen was used and for
assessment of antibodies against T. mutans, the 32 kDa

ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 61–62 61
Comparison Between Reverse Line Blot and Enzyme-linked Immunosorbent Assay in Diagnosis of Major Tick-borne Diseases D. A. Salih
et al.

Table 1. Cross tabulation of the results of reverse line blot (RLB) and Acknowledgements
enzyme-linked immunosorbent assay (ELISA) for Theileria parva,
Theileria mutans and Babesia bigemina This research was supported by the International Founda-
tion for Science, Stockholm, Sweden and Organisation of
RLB
Islamic Conference Standing Committee on Scientific and
T. parva T. mutans B. bigemina Technological Cooperation (COMSTECH), Islamabad,
Pakistan, through a grant (IFS grant 3765-1) to Diaeldin
ELISA Pos. Neg. Pos. Neg. Pos. Neg.
Ahmed SALIH. Supported in part by EU CA project
T. parva Pos. 53 19
Neg. 11 7
ICTTD-3 (no. 510561).
T. mutans Pos. 71 9
Neg. 8 2
References
B. bigemina Pos. 0 46
Neg. 0 44 Anonymous, 1983: Tick and Tick-Borne Disease Control. The
Sudan: Studies on Important Tick-Borne Diseases of Cattle.
Final Report, AG: CP/SUD/024/DEN, Rome.
protein ELISA test was applied while for B. bigemina the Bishop, R., B.K. Sohanpal, D.P. Kariuki, A.S. Young, V. Nene,
200 kDa antigen ELISA was used (Svanova, Uppsala, Sweden). H. Baylis, B.A. Allsopp, P.R. Spooner, T.T. Dolan, and S.P.
Morzaria, 1992: Detection of a carrier state in Theileria
parva-infected cattle by the polymerase chain reaction.
Results and discussion
Parasitology 104, 215–232.
Among 90 field samples from Central Equatoria State, Burridge, M.J., 1971: Application of the indirect fluorescent
Southern Sudan, ELISA reported more positive samples antibody test in experimental East Coast fever (Theileria
than RLB did (Table 1). The two tests positively recog- parva infection of cattle). Res. Vet. Sci. 12, 338–341.
nized 53 of the 90 samples as having T. parva antibodies El Hussein, A.M., A.A. Majid, and S.M. Hassan, 2004: The
and DNA, while seven samples were negative for T. parva present status of tick-borne diseases in the Sudan. Archs.
infection using the two techniques. The concordance of Inst. Pasteur Tunis, 81, 1–4.
Gubbels, J.M., A.P. de Vos, M. van der Weide, J. Viseras, L.M.
RLB showed 66.7% (60/90) relative to the results of
Schouls, E. de Vries, and F. Jongejan, 1999: Simultaneous
ELISA. With regard to T. mutans, there were 71 positive
detection of bovine Theileria and Babesia species by reverse
samples in RLB among 80 positive samples in ELISA,
line hybridization. J. Clin. Microbiol. 37, 1782–1789.
while two samples were negative in RLB out of 10 nega-
Kiltz, H.H., G. Uilenberg, F.F.J. Franssen, and N.M. Perié,
tive in ELISA. Subsequently, the concordances of RLB
1986: Theileria orientalis occurs in Central Africa. Res. Vet.
showed 81.1% (73/90) relative to the results of ELISA. Sci. 40, 197–200.
Among the 90 field samples, there were 46 samples that Salih, D.A., A.M. EL Hussein, U. Seitzer, and J.S. Ahmed,
reported positive by ELISA for B. bigemina antibodies, 2007: Epidemiological studies on tick-borne diseases of
while RLB did not detect any of these 90 samples as posi- cattle in Central Equatoria State, Southern Sudan. Parasitol.
tive showing concordance of 48.9% (44/90) relative to the Res., 101, 1035–1044.
results of ELISA. It has to be borne in mind that the Salih, D.A., S.M. Hassan, I.I. Julla, M.N. Kyule, K.-H. Zessin,
results of ELISA might represent previous infections, and A.M. EL Hussein, 2008: Distribution and application of
while that of RLB would not only reflect an active infec- ELISA for the seroprevalence of tick-borne diseases in
tion, but also a carrier status. Therefore, the selection of Central Equatoria State, Sudan. Transbound. Emerg. Dis. 55,
the test would depend on the specific aims of the study. 257–262.

62 ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 61–62