Prevalence and Molecular Diagnosis of Babesia ovis and Theileria ovis in Lohi Sheep at Livestock Experiment Station (LES), Bahadurnagar, Okara, Pakistan

Abstract

Babesia ovis and Theileria ovis are among the important and main etiological agents causing ovine babesiosis and ovine theileriosis, causing severe economic losses among sheep and goats. The aim of thee present study was to determine the prevalence and molecular diagnosis of B. ovis and T. ovis in Lohi sheep at Livestock Experiment Station Bahadurnagar, Okara, Pakistan. The prevalence of B. ovis and T. ovis was investigated in 200 Lohi sheep of mixed age and sex by PCR during 2011. The assay was employed using primers Bbo-F & Bbo-R, specific for a 549-bp fragment in B. ovis genomic DNA and primers TSsr 170F & TSsr 670R, specific for a 520-bp fragment in T. ovis genomic DNA. The animals were also screened for both haemoparasites through stained thin blood smears. Thirty two (16%), 48 (24%) and 26 (13%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites respectively, through microscopy. Sixty eight (34%), 73 (37%) and 42 (21%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites respectively, through PCR test. The results indicate the high sensitivity of PCR for surveying babesiosis and theileriosis and there is noteworthy prevalence of these diseases in sheep at an experimental station where environmental conditions are relatively controlled as compared to field conditions.

Headnote

Received 15Jun2013

Accepted 19 Sep 2013

Keywords

Babesia o vis,

Theileria o vis,

PCR,

Sheep,

Pakistan

Abstract

Background: Babesia ovis and Theileria ovis are among the important and main etiological agents causing ovine babesiosis and ovine theileriosis, causing severe economic losses among sheep and goats. The aim of thee present study was to determine the prevalence and molecular diagnosis of B. ovis and T. ovis in Lohi sheep at Livestock Experiment Station Bahadurnagar, Okara, Pakistan.

Methods: The prevalence of B. ovis and T. ovis was investigated in 200 Lohi sheep of mixed age and sex by PCR during 2011. The assay was employed using primers Bbo-F & Bbo-R, specific for a 549-bp fragment in B. ovis genomic DNA and primers TSsr 170F & TSsr 670R, specific for a 520-bp fragment in T. ovis genomic DNA. The animals were also screened for both haemoparasites through stained thin blood smears.

Results: Thirty two (16%), 48 (24%) and 26 (13%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites respectively, through microscopy. Sixty eight (34%), 73 (37%) and 42 (21%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites respectively, through PCR test.

Conclusion: The results indicate the high sensitivity of PCR for surveying babesiosis and theileriosis and there is noteworthy prevalence of these diseases in sheep at an experimental station where environmental conditions are relatively controlled as compared to field conditions.

Introduction

In Pakistan, livestock contributed ap- proximately 55.1% of the agriculture value added and 11.6% to the national GDP during 2011-2012 (1). Livestock sector's prospective role towards rural economic de- velopment can be gauged from the fact that 35M0 million rural populations are dependent on livestock, as every family has 2-3 cattle/ buffaloes and 5-6 sheep/goats, thus leading to derive 30-40% of their income from these an- imals (2). Sheep and goats rearing carry tre- mendous importance in rural economy partic- ularly for non-agricultural poor class of people (3). The population of sheep has been esti- mated as 28.4 million (M) in Pakistan and it yielded 0.037 M tons of milk for human con- sumption, 0.629 M tons of mutton and 0.043 M tons of wool during 2011-2012 (1). Punjab Province is the homeland for Lohi sheep and shares 24% of the total sheep population in the country (4).

Despite such a heavy population of live- stock in Pakistan, the production by these ani- mals is not as much as it could be. There are number of reasons for this discrepancy, e.g. poor breeding selection, management deficien- cies and prevalence of blood bom parasitic dis- eases (3). Pakistan, being a sub-tropical region of South Asia, has favorable environmental conditions for the growth of various ticks re- sponsible for transmitting different haemopara- sitic diseases in cattle and small ruminants. Small ruminant babesiosis and theileriosis de- velop in erythrocytes in peripheral blood and within the reticuloendothelial system.

Mostly three species of Babesia involved in causing the disease in ovine are; B. motas si, B. crassa and B. oris, (5). The disease caused by B. motassi may be acute and/or chronic (6), whereas B. crassa appears to have little or no pathogenicity (7).

B. oris is highly pathogenic especially in sheep and causes severe infection which is characterized by fever, anemia, icterus and hemoglobinuria with mortality rates ranging from 30 to 50% in susceptible host during field infections (8). It leads to significant losses among small ruminants due to its drastic ef- fect on hemobiotic system (9). Babesia species are also capable of causing diseases in humans (10) and is a significant public health threat due to emerging blood borne zoonosis in de- veloping countries like Pakistan. T. lestoquardi, T. seprata and T. oris are mostly involved among different species of Theileria that cause theileriosis in sheep. T. lestoquardi causes ma- lignant theileriosis in sheep and goats where as T. seprata causes low or nonpathogenic theiler- iosis (11). Theileria ovis is an important and main etiological agent of ovine theileriosis, causing severe economic losses among sheep and goats in tropical and subtropical areas in- cluding Pakistan (12-15). The disease is mani- fested by fever, progressive weight loss, de- crease in production and ultimately may lead to death.

Some workers studied the prevalence of both protozoans in areas which were different from the area which was selected for present study. Durrani et al. (12) has found Theileria spp. in 35% sheep blood samples in district Lahore. Iqbal et al. (13) has detected B. ovis in 50% sheep blood samples by PCR from southern Punjab (Pakistan). Similarly Durrani et al. (16) has detected T. ovis in 6% blood samples of sheep and goats through PCR, in Punjab and Khyber Pukhtoon Khaw Prov- inces of Pakistan. However, before present study, little or no work was done in Pakistan on molecular diagnosis of Babesia and Theileria species especially in Lohi sheep. These types of sheep maintained at Livestock Experiment Station (LES), Bahadurnagar, Okara showed clinical signs of Babesiosis and Theileriosis such as persistent fever, loss of body weight, jaundice, anemia and sudden death in young animals during the summer season in 2011- (May to July).

Traditionally the haemoparasitic diseases were diagnosed on the basis of clinical signs and microscopy. Both methods have the limi- tation of their inability to detect the subclinical (e.g. trophozoite stage of parasites in blood, which is very difficult to detect micro- scopically) and carrier animals. The carrier an- imals need to be brought in to limelight to know the exact status of haemoparasitic infes- tation in a herd so that disease control pro- gram can be implemented in a more rational way in order to enhance the productivity of the animals. The modem molecular tools of diagnosis have the ability to overwhelm the limitations of the conventional diagnostic techniques.

Keeping in view the advantage of PCR for identifying the exact species of Babesia and Theileria species and to know the prevalence of these haemoparasites, this study was initiated in Lohi sheep maintained at LES, Bahadurna- gar, Okara. In Pakistan, the previous studies on haemoparasitic infestation in sheep were carried out in some other breeds of sheep. There was no such type of study conducted in Lohi sheep previously.

Materials and Methods

A total of 1360 Lohi sheep of different age groups and sex were maintained at LES, Baha- durnagar, Okara. During the summer season, from May to July 2011, clinical signs such as persistent high fever up to 105 °F, loss of body weight, jaundice, loss of appetite, anemia and sudden death were observed especially in young lambs (1-9 months old). During these four months, a total number of 200 blood samples were collected from the ear veins of 200 animals of different age groups and sex showing signs and symptoms of both haemo- parasitic diseases. Thin smears were prepared, air dried, stained with Field's stain (Hema- Color(TM), Merck), and examined under oil im- mersion on a microscope (Digi 3, Labomed, USA) for the presence of intracellular Babesia schizonts and/or Theileria piroplasms based on morphology (17). At least 50 microscopic are- as per slide were searched carefully. Those blood smears were recorded as negative for Babesia spp. or Theileria spp. in which no schi- zonts or piroplasms were observed.

DNA isolation and polymerase chain reac- tions

A 5-ml blood sample was also collected from jugular vein of each of the above de- scribed 200 sheep and placed in 10 ml clean sterile vacutainers containing ethylenedia- minetetraacetic acid. They were brought to the laboratory and stored at -20 °C until used for DNA extraction for PCR amplification. Whole blood was used for the extraction of total genomic DNA using a commercially available DNA isolation kit (PureGene®, Cen- tra, Minnesota, USA) according to the manu- facturer's instructions. The nucleic acid was extracted, also from known negative samples (40 healthy sheep maintained at a private Live- stock farm at Okara). DNA concentration was determined spectrophotometrically at 260 Y]m and 280 Y]m and the number of genomic cop- ies was determined by using Minevra Biolabs kit "VenorGem®" and the samples were stored at -20 °C till further use.

Three sets of primers (Table 1) were used for PCR amplification of different gene seg- ments of both of the parasites. The first pair of oligo-nucleotide primer was used to amplify 549 base pair (bp) region of small sub unit rRNA (ssu rRNA) gene of B. oris (18). The second primer pair specific for Theileria spp. was used for the amplification of small subu- nit ribosomal RNA (srRNA) gene fragment (1098 bp) from Theileria species genomic DNA as described by Allsopp et al. (19). The third primer pair, described by Altay et al. (20), was used to amplify SSU rRNA gene fragment of 520 bp length, which is specific for Theileria oris genomic DNA. All PCR amplification re- actions, including control negative samples, were carried out in a final volume of 25 pi containing 4 pi lysate or 2 ng purified DNA as DNA template, 12.5 pi commercially available PCR master mix (Pyro Start(TM) Fast PCR Mas- ter Mix- 2X, Fermentas, Ontario, Canada), 2 U Taq DNA polymerase (Thermoscientific, Cali- fornia, USA) and 2 mM MgCh (Thermo scien- tific, California, USA). The primers were used at a concentration of 10 pmol pi"1 for B. ovis and 80 pmol pi"1 for Theileria spp. and T. ovis.

PCR was carried out in a DNA thermal cy- cler (PEQLAB Biotechnologie, GmbH, Erlan- gen, Deutschland) with following cycling pro- gram for B. ovis: 94 °C for 3 min, followed by 35 cycles at 94 °C for 1 min, 62 °C for 1 min and 72 °C for 1 min, with a final extension step at 72 °C for 5 min The amplification was carried with 30 cycles for Theileria species spe- cific primer set. After initial dénaturation at 94 °C for 3 minutes., each cycle involved déna- turation for 1 min at 94 °C, annealing for 1 min at 60 °C and extension for I min at 72 °C, with a final extension step at 72 °C for 5 min Cycling conditions for T. ovis were 96 °C for 3 min followed by 30 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 2 min with a final extension step at 72 °C for 10 min. Ten mi- croliters of the PCR products admixed with 6X DNA loading dye (TM) (Fermentas, Ontario, Canada) were sized by electrophoresis on a 1% agarose gel (Gene choice ®, Maryland, USA) (lh at 90 V) with 1-kb ladder (Fermen- tas, Ontario, Canada) as size marker. The gels were stained with ethidium bromide (HP 47.1, ROTH, Karlsruhe) (250 pg/ml @ one drop/100ml of 1% agarose gel) and analyzed in a UV trans-illuminator (Dolphin -Doc, Weal tec, NV, USA) for visualization of PCR products.

Results

The stained blood films revealed schizonts as teardrop-shape in pairs in red blood cells which were identified as Babesia, while piro- plasm appeared in oval and ring forms, detect- able inside the red blood cells and these were identified as Theileria (5, 17). Out of the 200 blood smears examined, 32 (16%) samples were positive for Babesia, 48 (24%) for Theileria while 26 (13%) were positive for mixed infec- tions with both parasites through microscopy. Data recorded for extracted DNA concentra- tion by spectrophotometrie analysis showed optical density values of 1.83 and 0.83 at 260 7]m and 280 Y]m wavelengths, respectively. Sixty eight (34%) out of 200 samples showed amplification of a 549-bp fragment (Fig la) with the primer pair Bbo-F and Bbo-R, specif- ic for B. ovis. Similarly 1098-bp fragment (Fig. lb) was generated with primer pair 989F and 990R specific for Theileria spp. in 78/200 (39%) samples while a 520-bp fragment (Fig. lc) was amplified with primer pair TSsrl70F and TSsr670R specific for T. ovis in 73/200 (37%) samples. Forty two (21%) samples showed mixed infection and were positive for both T. ovis and B. ovis. No such amplicon was detected in control negative samples. All sam- ples positive with microscopic examination of blood smears, were also positive with PCR test. The performance of both tests in terms of detection of positive cases has been com- pared in Table 2. Statistically (Z- test statistic), there was significant difference between the performance of PCR technique and micros- copy for the diagnosis of haemoparasites.

Discussion

Several studies in Pakistan revealed that B. ovis and T. ovis are endemic in sheep and goats in different parts of the country (12, 13, 15, 16, 21, 22). There are very few case reports about sheep being infested with these para- sites. This is the first molecular diagnostic re- port about the prevalence of B. ovis and T. ovis in Lohi sheep at LES, Bahadumagar, Okara, Pakistan. Iqbal et al. (13) observed 50% preva- lence of B. ovis in sheep in Southern Punjab through PCR which was higher as compared to the prevalence of B. ovis (34%) found dur- ing the present study. The difference in preva- lence rate between the two studies may be at- tributed to the difference in geographical are- as. Also the author of previous study has not mentioned the specific breed/s of sheep, which might be more susceptible to this dis- ease as compared to Lohi breed. Durrani et al. (12) reported 27.5% (55/200) prevalence of T. ovis in sheep through PCR at district Lahore, which was less than the prevalence of the same protozoan (37%) in the present study. Although climatic conditions of the two study areas are almost the same, yet the higher prev- alence of T. ovis in the present study cannot be compared with low prevalence reported in the previous study due to the facts that in present study the samples were collected in peak summer season which was ideal for tick infes- tation, as compared to the previous study in which the samples were collected in spring season, when the tick population was not as much as that was in summer season. Another reason for this discrepancy was the animal rearing type difference. In the present re- search, the samples were collected from inten- sive farming system at LES Bahadumagar, Okara as compared to the collection of sam- ples from individual animal keepings in the previous study. The prevalence for a mixed infection (21%) with T. ovis and B. ovis has also been observed during this study which is sup- ported by a previous study conducted by Inci et al. (23) who observed 1.7% (7/421) preva- lence of mixed infection for T. ovis & B. ovis in sheep in central Anatolia (Turkey).

The Lohi sheep which is an indigenous breed in Pakistan is recognized as relatively disease resistant breed as compared to con- temporary sheep breeds in this country. Dur- ing this study, it was noted that this type of sheep is susceptible to tick bom diseases. The environment at LES is relatively controlled as compared to the field conditions and tickicidals are being used regularly especially in the summer season. The results of this study are indicative of the fact that even a very few ticks can cause the spread of Babesiosis and Theileriosis. Although the disease prevalence recording in different age groups was not the main focus of this study, but it was observed that the disease was more prevalent in animals less than one year of age as compared to the animals having more than one year of age. This finding is in accordance to the findings by Ramzi et al. (24) and Iqbal et al. (13) who were of the view that probably the animals develop immunity against these parasites with the age. The disease was diagnosed in small mminants through symptoms and microscopy in earlier studies in Pakistan (15, 21, 22). Mi- croscopic detection methods are still the cheapest and fastest methods used to identify Babesia and Theleria parasites, although these have limited sensitivity and specificity. These techniques can detect for up to one infected erythrocyte per ten thousand cells, requiring the analysis of 100-200 fields, the equivalent to 0.5 pi of blood. It is an inexpensive tech- nique but requires an experienced person to differentiate species and is reliable only if the amount of parasites in the blood is high enough to be detected, which is usually only possible during acute cases (25). The obser- vation of paired intra-erythrocytic merozoites is indicative of infection but there are other stages of the parasite like the trophozoites, which present different forms and sizes de- pending on the species, and these make their detection difficult and time consuming. An exact differentiation between the parasites is crucial to understand their epidemiology. De- tection methods based on nucleic acid identi- fication and their amplification are the most sensitive and reliable techniques available to- day; they are fast, very specific and although most of them relay on sophisticated equip- ment (25). Recently, molecular techniques have become the preferred methods for diag- nosis of babesiosis and theileriosis, because these techniques are more sensitive and spe- cific than other conventional methods (20, 26).

In this study a molecular technique was used to diagnose parasitic infestation in Lohi sheep. Microscopy of blood smears once again proved to be less sensitive as compared PCR. Through the latter technique the parasites were identified and confirmed up to genus level. Morphologically only a few blood smears were identified up to species level. During this study, all the samples found posi- tive for haemo-parasites through microscopy, were also found positive for these protozoon through PCR, however the molecular tech- nique detected some additional cases (of Theileriosis) that were found negative with microscopy. The specificity of PCR product of these samples was confirmed when com- pared to the amplicon size of those samples which were found positive with both of the techniques. Five samples positive for Theileria infestation detected through PCR were not typed with T. oris specific primers. The proba- ble reason behind this might be the genome sequence difference (due to involvement of some other strain/s or species of Theileria) that needed additional primer pairs specific for them. This hypothesis can be confirmed by gene sequencing of SSU rRNA gene which was beyond the scope of this study. The re- sults of present study are in agreement with the findings of previous studies (12, 13, 16) who also compared microscopic examination test with PCR in small ruminants and found PCR to be a sensitive method for B. oris and T. oris diagnosis. Similar observations have also been made earlier (18, 20, 27-32), while comparing PCR with blood smear examina- tion.

Conclusion

PCR for the detection of B. oris and T. oris is specific and sensitive. The tests is suitable for tracing carrier animals and provide a quali- tative and validated measure that is useful in epidemiological surveys and follow ups for drug treatment in Lohi sheep. In addition, it would be useful while designing haemopara- sitic (ovine babesiosis and ovine theileriosis) control program in endemic areas. The study also revealed that Lohi sheep is very much susceptible to babesiosis and theileriosis. The prevalence rate of haemoparasitic infestation in sheep at LES cannot be disregarded. Special attention should be paid for the control of tick infestation to the animals in order to enhance the livestock productivity.

Acknowledgements

The authors are thankful to Director, Live- stock Production Research Institute, Baha- dumagar, Okara for providing necessary re- search facilities. Partial support for this re- search study was provided through the project of Government of Punjab titled "Up- grada- tion of research facilities at LPRI, Bahadurna- gar, Okara". The authors also gratefully acknowledge Mr. Sohail Ahmad (Research Officer, Statistics) for his valuable contribu- tion to statistical inferences. The authors de- clare that there is no conflict of interest.

References

References

1. Anonymous. Economic Survey of Pakistan. Fi- nance Division. Economic Advisors Wing. Ministry of Finance. Government of Pakistan, Islamabad; 2012. p. 29-30.

2. Anonymous. Economic Survey of Pakistan. Fi- nance Division. Economic Advisors Wing. Ministry of Finance. Government of Pakistan. Islamabad; 2011. p. 28-29.

3. Shahzad W, Munir R, Rana MY, Ahmad R, Khan MS, Akbar G, Ijaz M, Mehmood F. Prevalence, molecular diagnosis and treatment of Mycoplasma conjunctivae isolated from in-fectious keratoconjunctivitis affected Lohi sheep maintained at Livestock Experiment Sta-tion, Bahadurnagar, Okara, Pakistan. Trop Ani Health Prod. 2013; 45:737-742.

4. Anonymous. Pakistan Livestock Census. Statis-tics Division. Agricultural Census Organization. Government of Pakistan. 2006; p. 22.

5. Soulsby EYL. The Helminths, Arthropods and Protozoa of domestic animals. Bailliere & Tin-dall. London; 1986. p. 809.

6. Morel P. Tick-Borne diseases of livestock in Africa. In: Fischer MS, Ralph S, editors. Man-ual of Tropical Veterinary Parasitology. CAB International. Wallingford; 1989. p. 473.

7. FriedhoffKT. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Ana-plama spp. Parasitologia. 1997; 39: 99-109.

8. Hashemi-Fesharki R. Tick-borne diseases of sheep and goats and their related vectors in Iran. Parasitologia. 1997; 39: 115-117.

9. Radostits OM, Gay CC, HinchcliffKW, Con-stable PD. Veterinary Medicine. A textbook of the diseases of cattle, horses, sheep, pigs and goats. 10th ed. WB Saunders Co. London, New York, Philadelphia; 2007. 1482-1540.

10. Kim JY, Shin-Hyeong C, Hyun-Na J, Tsuji M, Sung-Ran C, Park J, Gyung-Tae C, Jung-Won J, Cheun H, Hyeong-Woo L, Young-Hee L, Tong-Soo K. First case of human Babesiosis in Korea: Detection and characterization of a novel type of Babesia sp. (KO1) similar to ovine Babesia. J Clini Microbiol. 2007; 45: 2084-2087.

11. Sayin F, Serpil N, Bayram AY, Ayse C, Zafer K. Epidemiological studies on sheep and goat Theileria infection. Ankara Uni Vet Fakultesi Dergisi. 2009; 56: 127-129.

12. Durrani AZ, Younus M, Kamal N, Mehmood N, Shakoori AR. Prevalence of ovine Theileria species in district Lahore, Pakistan. Pak J Zool. 2011; 43: 57-60.

13. Iqbal F, Ali M, Fatima M, Shahnawaz S, Zu-lifqar S, Fatima R, Shaikh RS, Shaikh AS, Aktas M, Ali M. A Study on prevalence and deter-mination of the risk factors of infection with Babesia ovis in small ruminants from southern Punjab (Pakistan) by PCR amplification. Para-site. 2011; 18: 229-234.

14. Esmaeilnejad B, Tavassoli M, Asri-Rezaei S. In-vestigation of hematological and biochemical parameters in small ruminants naturally in-fected with Babesia ovis. Vet Res Forum. 2012; 3: 31-36.

15. Naz S, Maqbool A, Ahmed S, Ashraf K, Ah-med N, Saeed K, Latif M, Iqbal J, Ali Z, ShafiK, Nagra IA. Prevalence of theileriosis in small ruminants Lahore -Pakistan. J Vet Ani Sci. 2012; 2: 16-20.

16. Durrani S, Khan Z, Khattak RM, Ali M, Hameed H, Taqddas A, Faryal M, Kiran S, Riaz M, Hussain S, Shiek RS, Ali M, Iqbal F. A comparison of the presence of Theileria ovis by PCR amplification of their ssu rRNA gene in small ruminants from two provinces of Paki-stan. Asian Pacific J Trop Dise. 2012; 2: 43-47.

17. Zajac AM and Conboy GA. Veterinary Clinical Parasitology, 7th ed. Blackwell Publishing Ltd. UK; 2000. 172-175.

18. Aktas M, Altay K, Dumanli N. Development of a polymerase chain reaction method for di-agnosis of Babesia ovis infection in sheep and goats. Vet Parasitol. 2005; 133:277- 281.

19. Allsopp BA, Baylis HA, Allsopp MT, Cavalier-Smith T, Bishop RP, Carrington DM, So-hanpal B, Spooner P. Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences. Parasitology. 1993; 107: 157-165.

20. Altay K, Dumanli N, Holman PJ, Aktas M. Detection of Theileria ovis in naturally infected sheep by nested PCR. Vet Parasitol. 2005; 127: 99-104.

21. Shahabuddin, Nawaz Y, Nawaz J, Nawaz M. Epidemiological study on the occurrence of natural babesiosis in sheep and goats of Balo-chistan, Pakistan. Proceedings of Parasitol. 2006; 42: 47-60.

22. Rashid A, Khan JA, Khan MS, Rasheed K, Maqbool A, Iqbal J. Prevalence and chemo-therapy of babesiosis among Lohi sheep in the Livestock Experiment Station, Qadirabad, Pa-kistan and environs. J Venomous Ani and Tox including Trop Dise. 2010; 16: 587-591.

23. Inci A, Ica A, Yildirim A, Duzlu O. Identifica-tion of Babesia and Theileria species in small ru-minants in Central Anatolia (Turkey) via re-verse line blotting. Turkish J Vet Ani Sci. 2010; 34: 205-210.

24. Razmi GR, Naghibi A, Aslani M R, Dastjerdi K, Hossieni H. An epidemiological study on Babesia infection in small ruminants in Mash-had suburb, Khorasan province, Iran. Small Rumin Res. 2003; 50:39-44.

25. Mosqueda J, Olvera-Ramirez A, Aguilar-Ti-pacamu G, Canto GL. Current advances in de-tection and treatment of Babesiosis. Current Medi Chem. 2012; 19:1504-1518.

26. Nagore D, García-Sanmartín J, García-Pérez AL, Juste RA, Hurtado. Identification, ge-netic diversity and prevalence of Theileria and Babesia species in a sheep population from Nortern Spain. Int J Parasitol. 2004; 34: 1059-1067.

27. Altay K, Dumanli N, Aktas M. Molecular iden-tification, genetic diversity and distribution of Theileria and Babesia species infecting small ru-minants. Vet Parasitol. 2007; 147: 161-165.

28. Altay K, Aktas M, Dumanli N. Detection of Babesia ovis by PCR in Rhipicephalus bursa col- lected from naturally infested sheep and goats. Res Vet Sci. 2008; 85: 116 -119.

29. Shayan P, Rahbari S. Differentiation of sheep Theileria spp. and Babesia spp. by polymerase chain reaction. J Vet Res. 2007; 62: 15-20.

30. Shayan P, Hooshmand E, Nabian S, Rahbari S. Biometrical and genetically characterization of large Babesia ovis in Iran. J Parasitol Res. 2008; 103: 217-221.

31. Bami MH, Khazraiinia P, Haddadzadeh HR, Kazemi B. Identification of theileria species in sheep in the eastern half of Iran using nested PCR-RFLP and microscopic techniques. Ira-nian J Vet Res. 2010; 11:262-266.

32. Dehkordi ZS, Zakeri S, Nabian S, Bahonar A, Ghasem,i F, Noorollahi F, Rahbari S. Molecu-lar and bio-morphometrical identification of ovine babesiosis in Iran. Iranian J Parasitol. 2010; 4: 21-30.