Abstract: This study was carried out on cattle to detect the seroprevalence of Theileria annulata and Babesia bigemina around the Sanliurfa province. A total of 191 randomly selected cattle were examined from selected locations for Theileria annulata and Babesia bigemina. Blood samples were collected from the cattle by jugular vene puncture to obtain sera for Indirect Fluorescence Antibody Test (IFAT). Thin blood smears were prepared from the punctured ear veins of each animal. The blood smears were stained with 5% Giemsa’s stain and examined microscopically at 100x magnification. In 19 of 191 cattle (9.94%) T. annulata and in 17 cattle (8.90%) Babesia sp. were observed. The sera were tested for the presence of antibodies to the T. annulata and B. bigemina by IFAT. Antibodies were detected against T. annulata in 138 (72.25%) and B. bigemina in 84 (43.97%) sera of the tested 191 cattle.

INTRODUCTION

Theileria and Babesia sp. are tick-borne haemoprotozoan parasites of vertebrates that have a major impact on livestock production, mainly cattle and small ruminants, in tropical and subtropical areas (Sevinc et al., 2001; Kaya et al., 2006; Ica et al., 2007).

The protozoan parasite Theileria annulata is the causative agent of tropical theileriosis and endemic in the area around the Mediterranean and reaches the Middle East and the Southern of Asia. Tropical theileriosis is a protozoan infection seen in cattle, buffola, zebu and bison. It causes severe infection especially in cattle. The parasite is transmitted from cattle to cattle by ticks of the genus Hyalomma. In Turkey, T. annulata is considered to be a major threat to the cattle breeding since the disease causes mortality and economic losses, particularly in crossbred cattle (Mimioglu et al., 1971; McCosker, 1981; Blood and Radostits, 1989).

Diagnosis of the disease is based on clinical findings and Microscopic Examination (ME) of blood and lymph node smears stained with Giemsa in acute cases. Frequently serological methods are employed in determining subclinical infections. Since morphological structures of piroplasm form Theileria sp. similar to each other, differential diagnosis of specific species is difficult (Aktas et al., 2001a; Vatansever and Nalbantoglu, 2002).

Studies focusing on prevalence of tropical theileriosis in Turkey routinely utilized ME of peripheric blood and lymph node smears (Goksu, 1970; Tuzer, 1981; Dumanli and Ozer, 1987). Recently, indirect fluorescence antibody test has been reported to be used for the same purpose (Eren et al., 1995; Sayin et al., 2002; Aktas et al., 2002; Dumanli et al., 2005).

Babesiosis is one of the more common diseases of farm animals worldwide and is gaining increasing attention as an emerging tick-borne zoonosis in humans. Bovine babesiosis, caused by the tick-transmitted protozoan Babesia bigemina, B. bovis and B. divergens, is considered one of the most frequent and important tickborne diseases of cattle worldwide. Babesiosis is considered to be another major threat to the cattle industry since it causes mortality and economical losses in cattle farms as well (Cakmak, 1987; Inci, 1992; Eren, 1993; Tanyuksel et al., 2002; Ica, 2004).

There are four Babesia sp. in cattle in Turkey, which are Babesia bigemina, B. bovis, B. divergens and B. major. The greatest economic losses occur due to B. bigemina and B. bovis. In many areas of Turkey both species of Babesia occur concurrently, transmitted by one vector, Rhipicephalus annulatus (formerly Boophilus annulatus). The vectors of B. bigemina are coincided with babesiosis in various regions of Turkey (Cakmak, 1987; Dincer et al., 1991; Inci, 1992; Eren, 1993; Aydin and Bakirci, 2007).

The diagnosis of babesiosis is done by microscopical examination of blood smears and observing of clinical symptoms. Microscopic diagnosis is easier in acute form than in subclinical infections. Consequently, various serological tests are used in the diagnosis of subclinical infections. Many serological techniques have been developed for the detection of antibodies against the piroplasms in the last decades (Blood and Radostits, 1989; Cakmak, 1987; Eren, 1993; Sevinc et al., 2001).

The objective of the present study was to determine the seroprevalence of Theileria annulata and Babesia bigemina in cows in and around Sanliurfa by microscopic examination and IFAT.

MATERIALS AND METHODS

Area of the study and animals: The study was conducted in Sanliurfa province located in the South-Eastern of Turkey. A total of 191 cattle were sampled between March to October 2008. Blood samples were collected from randomly selected healthy cattle, in 5 different locations of namely Central (44), Siverek (35), Viransehir (30), Akcakale (38), Birecik (44). The owners of the animals were questioned about animal management and age; then obtained information was recorded. In the present study, the majority of the cattle raised at small-scale farms (4-19 heads per farms). Of these, 127 were 1-2 years old and 64 were between 3-5 years old.

Sample collection: The thin blood smears prepared from ear capillaries were fixed in methanol for 5 min and stained with 5% Giemsa solution for 30 min and the presence of Theileria and Babesia piroplasms were examined microscopically. At least 50 microscopical areas were carefully examined for Theileria and Babesia sp. piroplasms under the oil immersion lens. The presence of even a single piroplasm was considered positive.

For serum samples, 10 mL blood was collected in plain test tubes by jugular vene puncture. After collection, blood samples were stored in ice box for 4-6 h. The sera were seperated from the blood within 24 h by centrifugation at 2500 rpm for 10 min. Collected sera were aliquoted in 2 mL labelled tubes and kept at -20°C, until analysed by IFAT as described (Pipano and Cahana, 1969).

Serological examination: The IFAT, using both the schizont and piroplasm stage of the T. annulata and Babesia sp. piroplasm stages as antigens, was used to examine serum samples for the presence of appropriate specific parasite antibodies. T. annulata and B. bigemina antigens and control sera (positive-negative) were prepared in the Parasitology Department, Faculty of Veterinary Medicine, Ankara University used to detect antibodies to T. annulata and B. bigemina. Anti-bovine IgG, FITC Conjugate was obtained from SIGMA (Cat.No. F-7887). Slides were examined in dark room following the IFAT procedure using a flourescein microscope (Zeiss) with Neoflaur objective (40x).

Statistical analysis: The χ²-test was applied to compare the rates of seropositivity between age groups, study sites and gender. Statistical significance was defined as p<0.05. All statistical analyses were performed using SPSS.

RESULTS AND DISCUSSION

Out of 191 smears examined microscopically, 19 (9.94%) were positive for T. annulata and 17 (8.90%) Babesia sp. piroplasms. Of all the smears, three showed a mixed infection with piroplasms of Theileria and Babesia sp.

As shown in Table 1, antibodies to T. annulata were found in 138 (72.25%) and B. bigemina 84 (43.97%) of 191 cattle sera based on IFAT test results. In this study, antibodies to T. annulata and B. bigemina were determined in five districts in Sanliurfa. The prevalence of antibodies to T. annulata and B. bigemina for the five districts is presented by Table 1. As shown in Table 1, the highest seroprevalence for T. annulata was observed in Siverek (88.57%) and the lowest seropositivity was found in Viransehir (56.66%).

Table 1:	Prevalence of antibodies to T. annulata and B. bigemina in cattle in districts of Sanliurfa by the IFAT and microscopic examination

The seropositivity rates for Birecik (68.18%) and Centre (63.63%) were found as similar. The difference between the rates according to districts was statistically significant for T. annulata (p = 0.011).

The highest seroprevalence for B. bigemina was observed in Viransehir (56.66%) and the lowest seropositivity was found in Siverek (28.57%). The seropositivity rates for Birecik (54.54%) and Akcakale (52.63%) were found as similar. The difference between the rates according to districts was also statistically significant for B. bigemina (p = 0.016).

A total of 127 sera samples from 1-2 years old cattle and 64 serum samples from 3-5 years old cattle were examined for T. annulata and B. bigemina. Antibodies to B. bigemina were found in 49 (38.58%) cows at the age of 1-2 and 35 (54.68%) at the age of 3-5. Besides, antibodies to T. annulata were found in 94 (74.01%) cows at the age of 1-2 and 44 (68.75%) cows at the age of 3-5. A statistically significant difference between the age groups was observed for antibodies B. bigemina (p = 0.034). On the other hand, there is no statistically significant difference in seropositivity rates for T. annulata among the age groups, although the seropositivity rate was higher for the 1-2 years age group than the other age group (p = 0.443).

Anti-T. annulata antibodies were detected in 76 (73.78%) of 103 male cows and 62 (70.45%) of 88 female cows. Although, the seropositivity rate for the male cows was higher than the rate for the female cows, the difference between the rates was not found statistically significant (p = 0.608). From the 44 (42.71%) out of 103 male cows and the 40 (45.45%) out of 88 female cows were detected anti B. bigemina antibodies. There was no statistically significant difference in seropositivity between gender (p = 0.704).

Tick-transmitted diseases such as babesiosis and theileriosis are economically important globally (Uilenberg, 1995). These animals have an important role in the transmission of the infection by ticks (Brown, 1990). The diagnosis of piroplasm infections are based on clinical findings and microscopic examination of Giemsa-stained blood smears. However, this method is not sensitive enough or sufficiently specific to detect chronic carriers, particularly when mixed infections occur. Serological tests are frequently used for diagnosis of latent infections. Nevertheless, it is difficult and time consuming to identify piroplasmic forms within the erythrocytes from carrier animals. Serological tests based on determination of antibodies developed against agent causing disease are applied (Pipano and Cahana, 1969; Vatansever and Nalbantoglu, 2002).

Aktas et al. (2001a) examined microscopically of peripheral blood smears for Theileria annulata and observed in 42.8% (285), 17.1% (292) and 34.8% (164) cattle in Elazig, Malatya and Tunceli, respectively. Sevinc et al. (2001) found T. annulata in 15 of 157 smears (9.55%) in Konya. Acici (1995) examined blood smears of 184 cattle suspected of theileriosis and found as positive, 26 (17%) for T. annulata. The prevalence of B. bigemina by the examination of Giemsa stained blood smears were found between 0.6 and 54.96% of cattle according to the studies performed in various regions of Turkey (Mimioglu, 1955; Goksu, 1959; Ozcan, 1961; Hoffman et al., 1971; Tuzer, 1981; Dincer et al., 1991; Dumanli and Ozer, 1987; Eren, 1993; Ozer et al., 1993; Acici, 1995; Aktas et al., 2001b; Sevinc et al., 2001; Ica et al., 2007).

In Turkey IFAT was first used to detect the seroprevalence of Babesia sp. and Theileria annulata in cattle by Cakmak (1987). Cakmak (1987) detected the seropositivity at the rates of 4.8 and 9.7% against B. bigemina and B. bovis in cattle in Beytepe village of Ankara, respectively. In the following studies, the seroprevalence of B. bigemina were found between 49.2 and 100% in cattle at various regions of Turkey (Cakmak, 1987, 1993; Dincer et al., 1991; Inci, 1992; Cakmak and Oz, 1993; Eren, 1993; Eren et al., 1995; Acici, 1995; Sayin et al., 1997; Aktas et al., 2001b; Inci et al., 2002; Karatepe et al., 2003; Dumanli et al., 2005; Ica, 2004; Sayin et al., 2004; Ica et al., 2007).

The prevalences of T. annulata were determined very high in different regions. The highest prevalences were found in Southeastern Anatolia, 91.40% and Central Anatolia, 92.65% (Dincer et al., 1991; Cakmak, 1993; Eren et al., 1995; Sayin et al., 1997; Aktas et al., 2001a; Vatansever and Nalbantoglu, 2002; Dumanli et al., 2005). High seroprevalence (81.20%) was found in Sanliurfa by Dumanli et al. (2005). Cakmak and Oz (1993) found T.annulata seropositivity as 10.68% in Adana. Acici (1995) carried out IFAT on serum samples collected from 76 cattle in Samsun; 48 cattle had antibodies to T. annulata. Kaya et al. (2006) found T. annulata 24 of 214 (11.21%) cattle in Hatay.

In this study, the prevalence of Babesia sp. and Theileria annulata by microscopic examination were respectively found to be 19 (9.94%) and 17 (8.90%). However, IFAT screening resulted with 138 (72.25%) T. annulata and 84 (43.97%) B. bigemina seropositive out of 191 animals.

CONCLUSION

The prevalence of T. annulata is widespread in cattle of Sanliurfa Province. Urgent measures such as anti-Theileria vaccines, chemotherapy, chemoprophylaxis and vector control should therefore be taken prevention of theileriosis.