Microsoft Word - افكار مسلم Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 1   ISOLATION OF THEILERIA AND BABESIA FROM GUT AND OVERY OF HARD TICKS: Hyalomma a. anatolicum IN BAGHDAD. Afkar M. Hadi A.M.A. Al- Amery * Iraqi Natural History Research Center and Museum - University of Baghdad. E-mail: afkar_hadi_iraq@yahoo.com ** College of Veterinary Medicine - University of Baghdad. ABSTRACT The application of thick blood smear technique based on Giemza- stain confirmed the presence of high rate of ticks infection 58.3% is revealed to endemic area of Theileriosis and Babesiosis in Iraq. Abdomen area (gut and ovary) of hard ticks: Hyalomma a. anatolicum revealed high rate of infection with Theileria sp.43% and Babesia sp. 15.2% . Babesia was recorded for the first time in Iraq from ticks. Females appeared positive smears more than males that total rate of infection (39) 54.1%. Distribution of infection rate in ticks was discussed with two periods of collection from May to October 2009. We concluded from this study that Hyalomma a. anatolicum tick at least one of Theileria &Babesia sp. Infective for cattle in Iraq; and this technique to be useful in identifying the species of protozoa in potential tick vectors. Keywords: Babesia, Theileria, hard ticks, Hyalomma anatolicum , salivary gland. INTRODUCTION Parasitic infections may be carried from one host to another by means of arthropod vectors. Many Theileria species cause diseases in cattle, of which one of the most economically important is East Coast fever (ECF), caused by T. parva. A more comprehensive review of the subject has been published by Irvin (1987).The distribution of the two most important cattle species, T. parva and T. annulata, correlates well with the distribution of their vectors. Theileria parva is transmitted predominantly by Rhipicephalus appendiculatus, which is ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Sept. 26 , 2011 . Accepted for publication April 19 , 2012 . Copyright © 2009-2012 by Diyala Agricultural Sciences Journal . Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 2   restricted to eastern, central and southern Africa; T. annulata is transmitted by several Hyalomma species, which are distributed widely over North Africa, southern Europe, the Middle East, India, southern Russia and China. There is virtually no overlap of these vectors and therefore the possibility of confusing these two parasites is low. (Conrad et al.,1987). Babesiosis, caused by infection with intraerythrocytic parasites of the genus Babesia, is one of the most common infections of free-living animals worldwide and is gaining increasing interest as an emerging zoonosis in humans. (Mary et al.,2000) . All babesial parasites described to date are transmitted by ixodid ticks to their vertebrate hosts. Recognized Babesia species of domestic animals by Kuttler(2004). The parasites replicate in the vertebrate hosts' red blood cells and are called piroplasms due to their pear-shaped appearance when within the infected host cells.(Kakoma and Mehlhorn,1993; Telford et al.,1993) In Iraq, the vectors of Theilerosis in cattle were: Hyalomma a. anatlicum 94.4%, Boophilus annulatus 4.4% ,Rhipicephalus sanguineus 1.4%.(Tarish ,1982). This study investigated the presence of some species of protozoa in the abdomen ( gut and ovary) of hard ticks: Hyalomma a. anatolicum as the widest distribution of cattles in Iraq. MATERIALS AND METHODS A total of 72hard ticks (9♂, 63♀) were collected from udder and perioneal region of cattles, their age between 1 – 3 years in Rashidia – Baghdad, for two periods: first period during May & July, second period during September & October 2009. All the ticks are diagnosed as Hyalomma a. analolicum by Iraqi Natural History Research Center and museum. Thick blood smear applied with simple modified as follow: 1- Inject tick with drop of Dextrose slain. 2- Cut anterior section of ticks, apply of blood drop on clean slide and leave it to dry. 3-Cut posterior section, apply of blood drop on clean slide and leave it to dry. 4-Fix the slides with absolute methanol for 5 min. 5-Dry the slides. 6-Put the slides in Giemsa- stain (10%) for 1 hour. 7-Wash the slides with tap water. 8-Examinated under light microscope with high power100X. Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 3   RESULTS AND DISSCUSION Microscopic of Giemsa-stained blood films revealed the presence of numerous Theileria and Babesia piroplasms for both sexes. Table 1. Table 1: Number of positive Ticks and percentage of infection ticks with Theileria and Babesia species. % of infected No. of ♀ infected % of infected No. of ♂ infected % of infected No. of positive ticks protozoa 44.4% 17.4% 28 11 33.3% _ 3 _ 43% 15.2% 31 11 Theileria sp. Babesia sp. 54.1%394.1%358.3%42total Significant difference p<0.05 between two sex.٭ The current study, described the application of thick blood smear technique based on Giemsa- stain to detect the possible presence of protozoa species in ticks. Giemsa-stained blood films confirmed the presence of numerous Theileria parasites. (Chae et al.,1998) The total rate of ticks infection 58.3% is high rate revealed to endemic area of Theileriosis and Babesiosis with or without appeared of clinical signs as in Iraq. Females appeared positive smears more than male as result for large quantities of blood that sucking from animals; that similar with Tarish( 1982) who recorded Theileria annulata 65.1% in female and34.9% in male of Hyalomma a, anatolicum from cattle. Abdomen area (gut and ovary) of ticks revealed high rate of infection with Theileria 43% and Babesia 15.2%, that mean reproduction process may be happened in gut of ticks in infected with Thieleria and migrated to ovary of ticks in infected with Babesia (Siegmund et al.,2010). Giemsa –stained blood film from Hyalomma a. anatolicum showing polymorphic Theileria parasite forms: Ring form (Fig.1), and multiple parasites are seen within hemolytic erythrocytes (Fig.2). Babesia forms appear as annular(Fig.3), pear- shaped(Fig.4) and bicornate form (Fig.5);The appearance of all these forms in ticks indicate that Iraq still now a day endemic area of Theileriosis & Babesiosis. Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 4   Fig.1:Ring form of Theileria sp. in ticks.100X. Fig.2: Multiple Theileria sp. are seen within hemolytic erythrocytes in ticks.100X. Fig.3:Annular form of Babesia sp. in tick.100X. Fig.4:Pear – shaped of Babesia sp. in tick.100X. Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 5   Fig.5:Bicornate form of Babesia sp. in ticks.100X The finding of Babesia sp. from abdomen of ticks is significant, because the definitive host is the tick ;Once ingested by an appropriate tick, gametes unite and undergo a sporogonic cycle resulting in sporpzoites. Transoverarial transmission has been documented for" large" Babesia spp. but not for the "small" Babesia, such as B. microti.(Zintl et al., 2003) . Distribution of infection with Babesia appeared highly significant differences between two periods. Table 2. Fig.6 Table 2: Distribution of infected ticks during two periods of 2009. % Second period (Sept. &Oct.) 38 ticks No. positive % First period (May & July) 34 ticks No. positive Protozoa 44.7% 26.3% 17 10 41.1% 2.9% 14 1 Theileria sp. Babesia sp. 71%2744.1%15Total Significant difference p< 0.01 between two periods.٭ The second period (September &October) of testing ticks revealed high rate of infection 71% with Theileria& Babesia sp. that may be because decreasing in the immune of cattle due to the peak of the infection with Theileriosis& Babesiosis in these months. Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 6   Fig.6: Distribution of infection of ticks during two periods 2009. There is no significant difference between two periods of infected with Theileria sp; that signal for endemic disease in cattle in Iraq. The mortality rate for tropical theileriosis can also vary from 3% to nearly 90%, depending on the strain of parasite and the susceptibility of the animals. (Pipano and Shkap,2000) There is highly significant difference between two periods of infected with Babesiosis sp; that mean the infection may be begun in first period (May & July) and reach the peak at second period (September &October). (Fig.1)We concluded from our results that Hyalomma a. anatolicum tick at least one Theileria &Babesia sp. Infective for cattle in Iraq; and thick blood smear with Giemsa technique to be useful in identifying the species of protozoa in potential tick vectors. ACKNOWLEDGEMENT The authors wish to thank Professor Mohammad K. Mohammad (Iraqi Natural History Research center and Museum, University of Baghdad) for diagnosis of hard ticks. REFERENCES Chae J. S.,J.M. Lee, O. D. Kwon, P.J. Holman, S.D. Waghela and G.G. Wagner. 1998. Nucleotide sequence heterogeneity in the small subunit ribosomal RNA gene variable(V4) region among and within geographic isolates of Theileria from cattle, elk and white-tailed deer. Vet. Parasit. vol. 75: 41–52. Conrad P. A., D. A. Stagg, J. G. Grootenhuis, A. D. Irvin, J. Newson, R. E. Njamunggeh, P. B. Rossiter and A. S. Young .1987. Isolation of Theileria parasites from African buffalo (Syncerus caffer) and 0% 20% 40% 60% 80% 100% first  period second  period Babesia Theileria PERIODS No. of  Positive Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 7   characterization with antischizont monoclonal antibodies. Parastl. 94: 413- 423. Irvin A. D. 1987. Characterization of species and strains of Theileria. Advan. Parasit. 26:145-197. Kakoma I. and H. Mehlhorn . 1993. Babesia of domestic animals. In J. P. Kreier (ed.), Parasitic protozoa, 2nd ed., vol. 7. Academic Press, San Diego, Calif. p. 141- 216. Kuttler K. L. 2004. BABESIOSIS. http://www.vet.uga.edu/vpp/gray_book/Handheld/bab.htm pp. 19 of 24. Mary J. H., A. D. Irma, R. T. Sam, J. K. Peter and H. P. David . 2000. Babesiosis .Clin. Microbio. vol. 13, No. 3 p. 451-469. Pipano E. and V. Shkap. 2000. Vaccination against Tropical Theileriosis. Ann. N.Y.Acad.Sci. 916: 484– 500. Siegmund O.H.,L.G. Eaton, W.W. Armistead, J.A.Henderson, T.L. Jones, J.W.McLean,and G.B. Schnelle. 2010. THE MERCK VETERINARY MANUAL. Tenth Edition. Telford S. R., A. Gorenflot,P. Brasseur, and A.Spielman. 1993. Babesial infections in humans and wildlife, p. 1-47. In J. P. Kreier (ed.), Parasitic protozoa, 2nd ed., vol. 5. Academic Press, San Diego, Calif. Tarish H. R.1982. A study of the role of the ticks in the epidemiology of the Theileria disease. MSc. Thesis submitted to the department of parasitology College of Veterinary Medicine University of Baghdad.p.73. WWW.merckbooks.com Zintl A.,G. Mulcahy ,H.E. Skerrett , S.M.Taylor ,and J.S. Gray .2003. Babesia divergens, a bovine blood parasite of veterinaryand zoonotic importance. Clin. Microbiol. Rev. vol.16:622-36. Hadi and Al-Amery Diyala Agricultural Sciences Journal, 4( 2 ) 1 – 8 ,2012 8   من القناة الهضمية والمبيض للقراد الصلب نوع هايلوما اناتولكم اناتولكم الثايليريا والبابيزيا عزل .في بغداد **عامر مرحم عبد العامري *أفكار مسلم هادي .جامعة بغداد -مركز بحوث ومتحف التاريخ الطبيعي * .جامعة بغداد –يطري كلية الطب الب ** المستخلص أظهر فحص المسحات السميكة لدم القراد المصبوغ بالكمزا انتشارالثايليريا والبابيزيا بنسبة كلية وطفيلي البابيزيا % 43وجود طفيلي الثايليريا بنسبة . تشير الى توطن المرضين في العراق% 58.3 للقراد الصلب نوع هايلوما اناتولكم ) والمبيضالقناة الهضمية (في منطقة البطن %15.2بنسبة أظهرت مسحات دم إناث القراد . اناتولكم، علما إن تسجيل البابيزيا من القراد ألول مرة في العراق ناقشت الدراسة توزيع اإلصابة خالل األشهر من %.54.1) 39(نسبة إصابة أعلى من الذكور بلغت نتج من الدراسة ان القراد الصلب نوع هايلوما اناتولكم نست .2009أيار إلى تشرين األول من عام اناتولكم ناقل حيوي لمرضي الثايليريا والبابيزيا في االبقار في العراق، والذي ظهر من فحص الدم . المصبوغ بطريقة الكمزا .البابيزيا، الثايليريا، القراد الصلب، هايلوما اناتولكم، الغدد اللعابية: الكلمات المفتاحية