1. Sudan J. Vet. Res. (2010). 25: 37-42.
With 1 table and 2 figures in the text.
37
Haemoparasites Identification in Two Dairy Cattle Farms in Khartoum State with
Reference of Babesia bigemina: molecular confirmation.
Ibrahim¹, A. M.; Geysen, D²; Ismail³, A. A.; Shadia A. Mohammed ¹
(1) College of Veterinary Science, University of Bahr el Ghazal. (2) Institute of Tropical
Medicine, Antwerp, Belgium. (3) College of Veterinary Medicine, Sudan University of Science
and Technology.
‫البحث‬ ‫ملخص‬
‫المنقول‬ ‫اض‬‫ر‬‫األم‬ ‫تعتبر‬‫ة‬‫ئيس‬‫ر‬‫ال‬ ‫المعوقات‬ ‫من‬ ‫اد‬‫ر‬‫الق‬ ‫اسطة‬‫و‬‫ب‬‫ة‬‫الماشي‬ ‫لتنمية‬‫ة‬.‫السودان‬ ‫في‬‫ي‬‫ر‬‫اج‬‫اس‬‫ر‬‫الد‬ ‫ىذه‬ ‫ت‬‫ة‬‫أثناء‬‫حدوث‬
.‫النفوق‬ ‫الى‬ ‫باألضافو‬ ‫ورقود‬ ‫الحميب‬ ‫في‬ ‫حاد‬ ‫نقص‬‫فحصت‬ ‫حيث‬69‫كانت‬ ،‫الخرطوم‬ ‫اليو‬‫و‬‫ب‬ ‫االلبان‬ ‫النتاج‬ ‫عتين‬‫مزر‬ ‫من‬ ‫دم‬ ‫عينو‬34‫عينو‬
(39.69‫موجب‬ )%‫ة‬‫ل‬‫طفيميات‬,‫الدم‬26( ‫منيا‬92‫البالغ‬ ‫األبقار‬ ‫في‬ )%‫ة‬‫و‬9(04.1‫في‬ )%.‫الصغيره‬ ‫العجول‬‫عدد‬ .02( ‫ه‬‫ر‬‫بق‬22.2‫تحمل‬ )%
‫طفيل‬‫يا‬‫ر‬‫الثال‬‫أما‬ .01‫الباقيو‬ ‫حالو‬(21.9‫م‬ ‫اصابات‬ ‫فتحمل‬ )%‫ختمط‬‫أىم‬ ‫و‬‫ا‬9( ‫عينات‬4.4‫بطفيل‬ ‫مصابو‬ )%‫يا‬‫ز‬‫البابي‬(‫و‬99.4‫المصابو‬ )%
‫بطفيميات‬‫و‬ ‫يا‬‫ز‬‫الباب‬‫األنابال‬‫امنو‬‫ز‬‫زمامت‬.‫عينات‬ ‫ثالث‬ ‫فحصت‬‫موجبو‬‫ع‬ ‫لتأكيد‬‫ه‬‫ر‬‫ت‬‫يا‬‫ز‬‫الباب‬‫يق‬‫ر‬‫ط‬ ‫عن‬‫الجزيئيو‬ ‫االحيبء‬ ‫تقنيو‬‫في‬ ‫متمثمو‬PCR-RFLP
‫فكانت‬‫ع‬‫نو‬ ‫من‬ ‫كميا‬Babesia bigemina.‫ف‬ ‫ممحوظ‬ ‫تطور‬ ‫سجل‬‫ي‬‫لعالج‬ ‫أدويو‬ ‫حقن‬ ‫بعد‬ ‫يضو‬‫ر‬‫الم‬ ‫األبقار‬ ‫صحة‬‫و‬ ‫يا‬‫ز‬‫الباب‬‫األنابالزم‬‫كما‬
‫نفو‬ ‫توقف‬‫االبقار‬ ‫ق‬‫ال‬‫حموب‬‫الناقل‬ ‫اد‬‫ر‬‫الق‬ ‫لجنس‬ ‫اع‬‫و‬‫ان‬ ‫وجود‬ ‫عدم‬ ‫من‬ ‫غم‬‫الر‬ ‫عمي‬ ‫انو‬ ‫الي‬ ‫اسو‬‫ر‬‫الد‬ ‫خمصت‬ .Boophilus‫ي‬ ‫أن‬ ‫ينبغي‬‫ف‬ ‫ؤخذ‬‫ي‬‫االعتبار‬
‫األ‬ ‫مرضي‬‫نابالزم‬‫و‬ ‫ا‬‫لم‬ ‫كأسباب‬ ‫يا‬‫ز‬‫الباب‬‫نفوق‬‫أ‬ ‫في‬ ‫ى‬‫األخر‬ ‫االقتصادية‬ ‫الخسائر‬‫و‬‫ال‬‫بقار‬‫ال‬‫حموب‬‫و‬ ‫الخرطوم‬ ‫الية‬‫و‬ ‫في‬‫ي‬‫من‬ ‫يد‬‫ز‬‫بالم‬ ‫وصى‬‫اسات‬‫ر‬‫الد‬‫عمي‬
‫ودور‬ ‫الناقل‬ ‫اد‬‫ر‬‫الق‬.‫الجانب‬ ‫ىذا‬ ‫في‬ ‫العاض‬ ‫الذباب‬
Summary
Tick-borne diseases are major constraints to livestock development in the Sudan. The
present paper reports on blood parasites in two dairy farms in Khartoum State. Sharp decrease
in milk yield, recumbency, as well as motilities of lactating cows were encountered in these dairy
farms. 96 cattle (61 from Shigla and 35 from West Soba areas) were examined. These consisted
of 75 milking cows and 21 calves (≤ 1 year old). Examination of the 96 blood smears revealed 47
positive samples (48.96%) for blood parasites. Thirty nine (83%) positive cases were adult
animals and the rest [8 (17.0%)] were calves. Thirteen adult cows (33.3%) were infected with
Theileria spp., 11 cows (28.2%) with Anaplasma spp. and two (5.1%) with Babesia spp. parasites.
The remaining 13 cows (33.3%) had mixed infections; five cases (41.7%) of Theileria and
Anaplasma spp. and eight cases (66.7%) of Babesia and Anaplasma spp. Babesia bigemina was
molecularly confirmed in 3 randomly selected cases using PCR-RFLP. Out of the 8 positive cases
of young calves, only one case (12.5%) was infected with Babesia spp., two cases (25%) with
Anaplasma spp. and three samples (37.5%) with Theileria spp. Two cases (25%) suffered a
mixed infection of Theileria and Anaplasma spp. The general health of the infected animals was
recorded following babesiosis and anaplasmosis treatment regimen. Moreover, mortility in the
adult cows stopped. It was concluded that despite the absence of Rhipicephalus (Boophilus) spp.
ticks in the study area, babesiosis and anaplasmosis should be considered to be among the causes
of mortility and other economical losses of dairy cattle industry in Khartoum State. Further
investigation into the available tick vectors and the role of biting flies in this aspect is
recommended.
Introduction
Exotic dairy cattle in the Sudan receive high attention, particularly in Khartoum State
as they constitute a major source of milk supply to the population of the state. Unfortunately,
ticks and biting flies are widespread in the area. Ticks and Tick-borne diseases (T&TBDs)

2. Ibrahim at al,29
play an increasingly negative role in animal health and livestock development plans
(Shommein 1979).
Indepth studies on T&TBDs in the Sudan, however, are still fragmentary and being complete.
In the Sudan, theileriosis was subjected more research activities (Hassan 1984; Elsadig 1993;
Kheir and Majid 1999; El Hussein et al. 2002; and Hassan and Salih 2009) compared to other
TBDs (Zessin and Baumann (1982); Morzaria and Pederson (1983); Sowar;( 2002) and Adam
(2005). A prevalence rate of 0.12% of babesiosis was reported in indigenous cattle in Sagadi
area (Paine 1983; Abdalla, 1984) during a field survey on TBDs in the Blue Nile and the
White Nile regions. Ahmed et al. (1987) reported babesiosis as one of the diseases which
causes high motility of exotic heifers imported to El Gezira State. B. bigemina was the
causative agent of the nine cattle cases of red water disease reported in Kassala region
(Mohamed and Yagoub, (1990).
In Khartoum State, anaplasmosis was occasionally diagnosed, but babesiosis was not
encountered in blood smears from cattels surveyed in Soba, Shambat and Hillat Hamad areas
(El sadig 1993). Similarly, Zein El aabdeen (1995) did not unable to diagnose any case of
babesiosis in cattle and sheep slaughtered in Khartoum State. However, 16(10.8%) and
39(50%) positive cases of bovine babesiosis were parasitologically and serologically detected,
respectively (Sulieman 2004) in Khartoum State.
The current situation of TBDs,other than theileriosis in Khartoum State was re-investigated.
The present communication reports on hemoparasite in two dairy farms in the Khartoum
State.
Materials and Methods
Study locations:
The two investigated Dairy cattle farms are located in El Shigla (El haj-Yosif),
Khartoum North and Soba West, Khartoum.
Animals:
The dairy cattle were cross bred lines with more than 60% Frisian blood. included in
this study 96 were lactating cows.
Blood Samples:
Ninety-six heparinized blood samples (61 from El Shigla and 35 from Soba) were
Submitted to the Parasitology laboratory, College of Veterinary Medicine, Sudan University
of Science and Technology, for hemoparasite examination. Thin dried fixed stained blood
smears were examined for blood parasites. Trypanosomes and microfilariae were also
investigated following blood centrifugation in a high-speed micro-haematocrit centrifuge
(Hawaksly-England). The buffy coat was examined for parasites following the method of
Murray et al. (1977).
Treatment:
Before collection of blood samples, the clinical cases in the two farms were treated
with Pubarvaqoun (Butalex®
) and different Antibiotics, several times; nonetheless there was
no good response and mortility continue. Following blood samples examination, Imidocarb
(Imizol®
), Diminazine diceturate (Berenil®
), Oxytetracycline (4 times at 3 days interval, IM)
as well as supportive treatment was administered to the infected animals. Dosage used and
regimen adopted were according to Cynthia et al (2005).
Molecular characterization and identification:

3. Blood Parasites Investigation in Two Dairy Farmsin Khartoum State 26
Three animals infected with Babesia species were rechecked and their blood samples
were spotted onto filter paper for molecular examination. These samples were investigated in
the Institute of Tropical Medicine (ITM), Antwerp, Belgium, using a single polymerase chain
reaction (PCR) - restriction fragment length polymorphism (RFLP) as described by Devos and
Geysen (2002) on the Ssu-rDNA locus. The extraction of DNA was done using saponin-PBS.
Restriction fragment length polymorphism:
Identification of suitable restriction enzymes was done with the aid of a computer
programme (Gene JockeyII). Nested products were digested with 10U/µg DNA Ddel enzyme
in NeB3 buffer on 6µl of amplified DNA in 15µl total volume. The reaction was left
overnight in a water bath at 37ºC.DNAfragments were thereafter separated on a
polyacrylamide gel by horizontal electrophoresis in 0.5x TBE buffer at 100V for 2.5h. A
100bp DNA ladder (MB1 Ferments, Lithuania) was used for fragment size determination. The
gel was stained using a commercial silver staining kit (Silver staining kit DNA plusone,
Pharmacia Biotech, Uppsala. Sweden).
Results
Case history:
Eleven of lactating cows died in El shigla area although several drugs including
Pubarvaqoun and/or antibiotics were administrated. A prolonged history of inappetence,
weight loss, sharp decrease in milk production and recumbency, particularly in Soba area was
reported before samples collection.
Microscopy examination:
Out of 96 animals screened, 34 were positives for piroplasms and 13 for Anaplasma
spp. indicating an overall tick-borne infection rate of 48.96%,( Table 1). Out of 47 positive
blood smears, 32 were due to a single infection Theileria spp. [16 (16.67%)], Anaplasma spp.
[13 (13.54%)] or Babesia spp. [3 (3.13%)]. The other 15 blood samples had mixed infection.
[8 (8.33%) Fig.1] and Theileria spp. with Anaplasma spp. [7(7.29%)]. No trypanosomes or
microfilariae were demonstrated.
Table 1: Prevalence rate of blood parasites in two dairy farms (n=96).
S.
No.
Haemoparasite Number infected (%) Prevalence
(%)Adult cows Young ≤ 1y
1 Theileria spp. 13 (13.54%) 3 (3.13%) 16.67%
2 Anaplasma spp. 11 (11.46%) 2 (2.08%) 13.54%
3 Babesia spp. 2 (2.08%) 1 (1.04) 3.13%
1 Theileria & Anaplasma spp. 5 (5.21%) 2 (2.08%) 7.29%
2 Babesia & Anaplasma spp. 8 (8.33%) 0 (0.00%) 8.33%
Total 39 (40.63%) 8 (8.33%) 48.96%
47
Macropathology:
A good progress in the general health and milk production of the treated animals was
improved following the adoption of babesiosis and anaplasmosis treatment regimen of
Cynthia et al (2005). Moreover, mortality of adult lactating cow.

4. Ibrahim at al,31
Fi. 1: Giemsa’s stained blood smear showing Babesia spp and Anaplasma spp. Mixed
infection.
1 2 3 4 5
Fig. 2: RFLP restriction enzyme analysis using Ddel enzyme on 18S-rDNA from
samples and fractionated on 10% PAGE gel, stained with silver. (Lanes 1, 2
and 3) Babesia bigemina. (Lane 4) Anaplasma margenale and (lane 5) 100bp
DNA ladder.
Discussion
Based on the absence of soft tick Boophilus tick vector in Khartoum State, occurrence
of bovine babesiosis in the State is currently not documented. The present study, reports the
first record of B. bigemina infection in dairy lactating cattle from Khartoum State. The PCR-
RFLP assay confirmed the investigation results. This study suggests high prevalence of
Babesia bigemina infection in Khartoum State. Similar results was reported serologically
(Sulieman 2004). This case of babesiosis had several unusual features. Firstly, it is fatal, and
occurred in Khartoum State where the presence of Boophilus tick is not documented, this is in
sharp contrast with the reported cases of bovine babesiosis (Paine 1983; Morzaria and
Pederson 1983; Ahmed et al. 1987; Mohamed and Yagoub 1990). Secondly, the prevalence
was high in contrast to the findings of El Sadig (1993) and Zein El Abdeen (1995). The
Babesia
Anaplasma marginale
sp
Anaplasma centrale
sp

5. Blood Parasites Investigation in Two Dairy Farmsin Khartoum State 30
possibility still holds that anaplasmosis and babesiosis were prevalent, but they were
commonly confused with theileriosis as it is the most prevalent tick-borne disease in the area.
Boophilus spp., (Soulsby 1986; Friedhoff 1988), the principal vectors of B. bigemina, was not
identified in the investigated area. The possibility of bringing some infected milking cattle
from the enzotic areas such as the White Nile State cannot be overruled. Other tick vectors
such as Rhipicephalus evertsi as well as biting flies may be responsible for distributing the
disease to these susceptible animals. Mixed infection of Babesia and Anaplasma (Fig.1)
increases the severity of the disease and in most cases ends fatally. Moreover, the misleading
belief that Khartoum is a Babesia free zone, has resulted in maldiagnosis of babesiosis and
concurrently led to application of different treatment regimen. Calves infected with Babesia,
in the present study were more than 9 months old. Similar observations were reported by
Sulieman (2004). Three parasitologically positive cases which were selected for molecular
identification were caused by Babesia bigemina. Disease due to B bigemina develops more
rapidly compared to B. bovis (Soulsby 1986).Exotic cattle breeds are highly susceptible to
infection with Babesia species, besides high mortality (Soulsby( 1986).
This study confirms PCR based technique, and for the first time, the occurrence of
Babesia bigemina in lactating cattle in Khartoum State.
Acknowledgements
We are indebted to the colleagues for facilitating this work: Dr. Badria Aamer and Dr.
Abu-Obaida. We are also grateful to ITM, Belgium, for molecular identification and our
special thanks are due to Lieve Vermeiren.
References
Abdalla, H. M. (1984). Studies on Babesia bigemina in cattle in Northern Sudan. M.V.Sc.
Thesis, Faculty of Veterinary Science, University of Khartoum, Khartoum, Sudan.
Adam, M. E. (2005). Prevalence of blood parasites in domestic animals in South Darfour
State of Sudan. M.V.Sc. Thesis. Faculty of Veterinary Medicine, University of Khartoum,
Khartoum, Sudan.
Ahmed, F. A.; El Agab, U. A. and Ismail, A. M. (1987). Vet. Quarterly. 9(2): 177-179.
Cynthia, M.; Kahn, B. A. and Scott Line, M. A (2005). The Merck Veterinary Manual. 9th
edn. Merck & Co; Inc. White House Station, N.J; U.S.A. Pp. 21-26.
Devos, J. and Geysen,, D. (2002). Vet Parsitol. 10 125 (3-4): 237-49.
El Hussein, A. M.; Majid, A. M. and Hassan, S. M. (2002). Archs. Inst. Pasteur
Tunis, 81: 1-4.
Asha A. El Sadig. (1993). Survey on prevalence of blood protozoa in dairy cattle in
Khartoum State. M.Sc. Thesis. Faculty of Veterinary Science, University of
Khartoum, Khartoum, Sudan.
Friedhoff, K. T. (1988). Transmission of Babesia. In: M. Ristic, (ed). Babesiosis of
Domestic Animals and Man, CRC Press, Boca Raton, Florida, USA, Pp23-52.
Hassan, S. M. (1984). Ticks and tick-borne diseases and Animal Wealth Development
in the Sudan (in Arabic). The proceeding of 11th
Veterinary Conference of Animal
Wealth in Eastern Sudan, Kassala. Part1. Pp. 165-168.
Hassan, S. M. and Salih, D. A. (2009). Bibliography with abstracts, Ticks and Tick-borne
Diseases in the Sudan. 1st
edn. published by The Central laboratory, Ministry of
Science and technology, Khartoum, Sudan.
Kheir, S. M. and Majid, A. M. (1999). Sudan J Vet. Sci Anim. Husb., 38(1&2): 137-142.

6. Ibrahim at al,31
Mohamed A. A. and Yagoub I. A. (1990). Trop. Anim. Hlth. Prod. 22(2):123-125.
Morzaria, S. P. and Pederson, V. (1983). Tick and Tick-borne disease control in the
Sudan. Studies on important tick-borne diseases in cattle. AG:
GCP/SUD/024/DEN. Technical report 2.Food and Agriculture
Organization of the United nations, Rome.
Murray, M.; Murray, P. R. and Mc Intyre, W. I. M. (1977). Acta Tropica, 27: 384-386.
Paine, G. (1983). Tick sand Tick-borne diseases control, the Sudan: Epizootiology. AG:
GCP/SUD/024/DEN. Technical Report 3.Food and Agriculture Organization of the
United Nations, Rome.
Shommein, A. M. (1979). Bull. Int. Epiz. 91: 425-431.
Soulsby, E. J. L. (ed.). (1986). Helminths, Arthropods and Protozoa of Domesticated
Animals. 7th
edn, Bailliere Tindall London. UK. Pp.761; 461-462.
Sowar, A. E. (2002). Epidemiology and ecology of ticks and some tick-borne diseases in
Kadogli and Dilling provinces Southern Kordofan State, Sudan. M.V.Sc. Thesis.
Faculty of Veterinary Medicine, University of Khartoum, Khartoum, Sudan.
Sulieman, T. A. (2004). Study on the epidemiology of ticks and tick-borne diseases in man
and cattle in Khartoum State. PhD Thesis. Faculty of Veterinary Medicine, University
of Khartoum, Khartoum, Sudan.
Urquhart, G. M.; Armour, J.; Duncan, J. L.; Dunn, A. M. and Jennings, F. W. (1996).
Veterinary Parasitology. 2nd
edn.-Blackwell Science. Pp. 307.
Zein Elaabdeen, S. M. (1995). Economic significance of piroplasmosis in cattle and sheep
slaughtered in Khartoum StateAbattoirs. M. T. A. H. Dissertation. Faculty of
Veterinary Science, University of Khartoum, Khartoum,Sudan
Zessin, K. H. and Baumann, M. P. O. (1982). A Report on the livestock diseases survey in
Bahr El Ghazal Province. Technical cooperation Germany/Sudan, GTZ, Berlin May
1982.