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��ࡱ�>��	mo����l��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������	��0jzbjbj��	;~x�x�sA�������&&i$i$i$i$i$����}$}$}$8�$�A%4}$�28u%:�%�%�%�%�&�&�&%2'2'2'2'2'2'2�4��72'2Qi$�&�&�&�&�&'2i$i$�%�%�x2�(�(�(�&Fi$�%i$�%%2�(�&%2�(�(V-@�-�%����p�vk!b�}$�&FQ-2�20�2]-T�7*'��7�-�-�7i$�-L�&�&�(�&�&�&�&�&'2'2�(�&�&�&�2�&�&�&�&���������������������������������������������������������������������7�&�&�&�&�&�&�&�&�&&	/#:	Seroprevalence of Theileria equi and Babesia caballi infection in horses in Iran

V. Abedia, G. R. Razmia,*, H. Seifib, A. Naghibia
a Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad
b Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of  Mashhad




Present address of Corresponding Author*:
G.R. Razmi
Ferdowsi University of Mashhad, Department of Pathobiology, Faculty of Veterinary Medicine, P.O. Box: 91775-1793
Tel: +98 511 8788944
Fax: +98 511 8763853
 HYPERLINK "mailto:razmi@um.ac" razmi@um.ac.ir







Abstract
Equine piroplasmosis is a hemoprotozoan tickborne disease with worldwide distribution, caused by Theileria equi and Babesia caballi. The aim of this study was to investigate the seroprevalence of T. equi and B. caballi infection in horses in the North Khorasan Province of Iran. Association between seropositivity of piroplasm infections and host-related factors include age, gender and activity was also analyzed. Blood samples were collected from 100 apparently healthy Turkoman breed horses (Equus caballus) in fourteen villages  from June to August in 2011. First, blood smears were prepared and stained with Geimsa method and then  serum samples were tested by indirect immunofluorescent antibody test (IFAT). T. equi was microscopically detected in five blood smears. The results were  also showed that 53 (53%) of samples were seropositive for piroplasm infections. Antibodies against T. equi was found in 48 (48%) of samples while B. caballi and dual infection in 2 (2%) and 3 (3%) of samples , respectively. Any siginificant difference was not obsereved between the sereopravalence of piroplasm infections with host-related factors in horses. This is the first report on the detection of the T. equi and B. caballi infection using IFAT in Iran, It was concluded that seroprevalence of T.equi infection is higher than B.caballi infection in Turkoman breed horse in Iran.
Keywords: Theileria equi, Babesia caballi, Indirect immunofluorescent antibody test, horse 







1. Introduction
Equine piroplasmosis is a tickborne hemoprotozoan disease of horses, mules, donkeys and zebras. It is caused by obligate intracellular parasites of the phylum Apicomplexa, include Theileria equi (formerly known as Babesia equi) and Babesia caballi[1,2].
The disease is characterized by fever, anemia, icterus, hepatosplenomegaly, bilirubinuria and hemoglobinuria . Peracute, acute, subacute and chronic forms of disease have been reported in horses. Most clinical cases is caused by T. equi and infections with B. caballi tend to be inapparent. Carrier horses may show clinical disease under stress condition 
Acute disease uncommon in endemic areas but introduction of infected equines into disease-free areas cause acute disaese and death, therefore control strategies in disease-free areas are directed against importation of infected equines [3-6]. 
Detection of Theileria and Babesia parasites carry out with direct or indirect methods. Direct methods including  microscopical and molecular methods and indirect methods accomplish by detection of antibodies against Theileria spp or Babesia spp in serum using serological methods. Examination of blood samples and identification of causative agent by stained blood smear is possible in acute phase of infection but it is difficult to detect parasites in blood smear of carrier horses with low parasitemias [7]. 
Among serological techniques, IFAT and ELISA have been recommended to detecte piroplam infections in international trade of horse[8]. 
Although ,B. caballi and T. equi were reported in horses of Iran [9,10], but, There are not any epidemiological study about piroplasm infection in the horses. The aim of this study was to determine the seroprevalence of the T. equi and B. caballi infection in Turkoman breed horses in Iran and evaluation the relationship between risk factors with seropositivity.


2. Matrials and methods
2.1. Field study area
This study was carried out in the North Khorasan Province, Iran. The province is located in northeastern of Iran between 36�372 -38�172  N latitudes and 55�532 -58�202  E longitudes with an area of more than 28,400 km2. It is situated next to the north eastern border of Iran, level with the southern caspian sea and south of Turkmenistan. This province has mountainous areas and receives about 250 mm of rainfall annually.
Turkmen tribes settled in parts of this province and Turkoman horse rearing common in this areas (Fig.1). Fourteen villages were selected for sampling. The villages have placed on border between Iran and Turkeminstan. Turkoman breed horse in Iran is similar to well-known Akhal-Teke breed in Turkmenistan. Turkoman breed horse bred in the north of Iran and east of Caspian sea [11,12]. 
2.2. Sampling
Blood samples collected from June to August in 2011. First, the data includhng  age, gender, activity were recorded for each horse. Then, the blood samples were taken from jugular vein using syringe and collected in plain tubes. The blood tubes were kept cool in transit to laboartory. Clotted blood was centrifugated and seperated sera were stored at -20�C until the time of serological examination.  
2.3. IFAT test
Antibody against T. equi and B. caballi infections were dectected by IFA IgG Antibody commercial kits according to manufacturer s instructions for T. equi antibody (Fuller Laboratories, Fullerton, Callifornia, USA) and for B. caballi antibody (MegaScreen, Horbranz, Austria). Sera were considered as positive if they showed fluorescence at dilution of 1:80.

2.4. Statistical analysis
Host-related Factors such as type of infection, age, gender and activity analyzed by chi-square test for any significant relationship.
3. Results
T. equi infection was microscopically detected in 5 (5%) blood smears with low parasitemia (approximately 0.001-0.003%).
Serological examination was revealed that 53 (53%) horses had antibodies against piroplasm infection.  Antibodies against T. equi, B. caballi and mixed of them were detected  in 48 (48%), 2 (2%) and 3 (3%) of seropositive horses, respectively (Table 1).
Any significantly different were not observed between the  seropositivity rates of T.equi and B.caballi  and  risk factors such as age, genders and activity groups in sampled horses (P>0.05) (Table.2).
4. Disscusion
In this study, T. equi infection were  microscopically detected in 5 (5%)  blood samples with low parasitemia, while  53% of horses were seropositive for piroplasms infection using IFAT. The results confirmed the low sensitivity of the microscopical examination in comparison with serology methods. So far, IFAT has been applied to differentiate T. equi and B. caballi infections and some studies were shown  that the sensitivity and specificity  of IFAT for T. equi and B. caballi is 89.2% ,92% and 96.6,95%, respectively [16,17].
Antibodies against T. equi, B. caballi and mixed of them  were detected  in 48 (48%), 2 (2%) and 3 (3%)  of seropositive horses, respectively.  Seropositivity rates of T. equi and B. caballi infections were reported  10.4 % and 7.5% in Saudi Arabia [21], 77.1 and 11.4% in Kuwait [22], 97.7 and 40.9% in Oman [23], 12.8 and 9.6% in Turkey [24] by IFAT method  and 16.21 and 0.83% in Turkey [25], 32.45 and 15.2% in United Arab Emirates [26], 14.6 and 0% in Jordan [27] by ELISA method , respectively. These serological studies confirmed that T. equi infection is more prevalent than B. caballi in Middle East countries.
In the present study, the  differences of seropositivity rate of  T. equi infection in  different age groups  were not significant.  The results were along with  the similar studies in Turkey [24,28], Italy[31], Israel [30,32], Switzerland [33] and  Venezuela [34] that were shown the age was not a risk factor in seropositivity rate in horses. This finding was  in contrary with other studies  that done in Mongolia[35] and Greece [13].
A  mare with her foal (one month old)  were also seropositive against T. equi infection.  The foal was healthy without any clinical sign. It seems  the  antibodies against T. equi can be transfered from mare to foal via colostrum. The antibodies in colostrum  is usually decrease after 63-77 days post foaling [36]. 
The difference of serporevalence in male and female horses were not statically significant. This finding is agreed with the obtained results in Turkey [24], Israel[32], Switzerland [33], Greece [13], Venezuela [34]and Trinidad[37] but disagreed with other studies in Israel [30]and Mongolia [35] that were showed a significant relationship between seroprevalence of piroplasm infections with sex factor. 
A non- significant differences  was obtained  between the seroprevalence of piroplasm infections in horses. The iranian horses with different activity are grazing in the pasture  during  spring and summer and the chance of tick infestion is high .Therefore, the occurrence of piroplasm infection is the same in horses with diferent activity .  A study was shown that the horses with reduced exposure to ticks have low infection rates of piroplasm infection [31].  Some studies were shown that the type of activity of horses with different health mangment significantly impacted the seroprevalence rate of infection [13.25]. During our study, all sampled horses were apprently health without clinical signs. The reason  of finding was unclear It may be related to  the breeding horse . The resistance of Turkoman breed horse to piroplasms infection  has not been studied yet. To the knowledge of the authors, this is the first study of seroprevalence of piroplasmosis in Turkoman horse in Iran . The findings of this study showed that equine piroplasmosis is endemic in the North Khorasan province. Although clinical signs of disease are rare, but T. equi infection is more prevalent than B. caballi infection. 

Acknowledgements
We thank Dr. Abdoljalil ghiadi the manager of Turkoman horse breeding farm for his hospitality and aid for sampling, Mohammad Abedi and Dr. Ali Sarani for their aid for sampling and data collection.

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36. Kumar S, Kumar R, Gupta AK, Dwivedi SK. Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares. Vet Parasitol 2008;151:80-5.
37. Asgarali Z, Coombs DK, Mohammed F, Campbell MD, Caesar E. A serological study of Babesia caballi and Theileria equi in Thoroughbreds in Trinidad. Vet Parasitol 2007;144:167-71.








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