Introduction

Ovine theileriosis in China was first reported by Yang et al. (1958). The disease was called Zado by the Tibetan people, and has existed in Gannan for more than 100 years. The causative agent of theileriosis of small ruminants in China was shown to be a new as yet unidentified parasite, which was referred to as Theileria species (China) (Schnittger et al. 2000). This parasite is transmitted by Haemaphysalis qinghaiensis and often occurs in mixed infections with other parasites (Yin et al. 2002a, b).

Gannan Tibet Autonomous Region is located in the southwestern part of Gansu Province in the northeastern part of Qingzang Plateau and the boundary region of Gansu, Qinghai and Sichuan Province. It represents the largest and most important stock-raising region of Gansu Province, with 1,700,000 sheep and goats, more than 75% of which are Tibetan sheep that are grazed in grassland areas. The goats are reared in the agricultural and semi-agricultural areas where the altitude is less than 3,000 m (Guo et al. 2002). Many of these small ruminants suffer from Theileria challenge each year, resulting in high economical losses.

This study represents the first that uses the enzyme-linked immunosorbent assay (ELISA) technique to widely survey ovine theileriosis in one of its epidemic areas in China.

Materials and methods

Serum samples

The 1,165 serum samples were collected from Biandu and Yangyong districts of Lintan County, Kache, Azitan and Wanmao districts of Zhuoni County and Ganjia, Zhayou and Madang districts of Xiahe County from July 2004 to June 2005 in Gannan Tibet Autonomous Region, Gansu Province (Table 1). Moreover, serum samples from an unchanging stable flock of 44 goats in Biandu district of Lintan County were collected at the end of every month from July 2004 to June 2005. Positive sera and negative sera were prepared as described by Gao et al. (2002).

Table 1 Positive rate of anti-Theileria sp. (China) antibodies in seven districts of Gannan Tibet Autonomous Region
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ELISA for the detection of ovine theileriosis

The antigen for performing ELISA was prepared from piroplasms of Theileria sp. (China) as described by Gao et al. (2002). Antigen was coated at a final concentration of 2.5 μg/ml in 100 μl carbonate buffer (0.05 M carbonate/bicarbonate buffer, pH 9.6) into each well of a 96-well microtitre plate (Nunc, Denmark) and incubated overnight at 4°C. All subsequent incubation steps were preceded by washing three times with phosphate-buffered saline (137 mM NaCl, 2.67 mM KCl, 3.2 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2) containing 0.1% Tween-20 (PBST). Blocking buffer (0.5% fish gelatine in carbonate buffer (pH 9.6) at a volume of 200 μl per well was added and incubated for 1 h at 37°C. Then, 100 μl of diluted serum samples (1:200 in PBST) were added and incubated at 37°C for 1 h. Horseradish peroxidase-conjugated rabbit anti-goat IgG (100 μl, diluted 1:40,000, Sigma-Aldrich, USA), was added followed by an incubation for another 1 h at 37°C. Lastly, 100 μl substrate (0.34 mg/ml orthophenylenediamine in phosphate/citrate buffer, pH 6.0, containing 0.05% hydrogen peroxide) was added to each well and allowed to develop for 30 min at room temperature. The reaction was stopped by adding 50 μl 1 M H2SO4, and the optical density (OD) values was read using the bichromatic differential absorbance of wavelengths 492 and 630 nm in an ELISA reader (Stat Fax 2600, USA).

Percentages were calculated using the following formula: %OD = (mean(sample OD) − mean(standard negative sera OD))/(mean(standard positive sera OD) − mean(standard negative sera OD)) × 100. If the values of the mean(standard positive sera OD)/mean(standard negative sera OD) were above 2, the result was valid. Values below 15% were rated as negative, and values above 15% as positive.

Detection of small ruminant piroplasms by reverse line blot

Analysis by reverse line blotting (RLB) was performed as previously described (Schnittger et al. 2004). Briefly, after extraction from blood samples using a genomic deoxyribonucleic acid extraction kit (Quiagen), the hypervariable region 4 of the 18S ribonucleic acid gene was amplified using primers RLB-F and RLB-R. RLB was performed using controls and probes for Theileria and Babesia species known to infect small ruminants (Schnittger et al. 2003, 2004).

Results

To verify the detection of ovine theileriosis, results obtained using the ELISA were compared to results using RLB in a total of 38 samples from the May collection and from 40 samples from the December collection of the stable flock. RLB analysis detected only Theileria sp. (China 1) and Theileria sp. (China 2) (Schnittger et al. 2003); no Babesia species were detected in the samples. Thus, the ELISA system was judged suitable for the detection of ovine theileriosis.

The results of the analysis of the 1,165 sera samples collected from seven different districts are summarized in Table 1. The analysis showed that the seroprevalence of anti Theileria spp. antibodies ranged from 27.8 to 83.3% in different geographical areas in Gannan Tibetan Autonomous Region and that the average was 70.1%. The data are in accordance with previous investigations based on determination of parasitemia (Luo and Yin 1997; Guo et al. 2002) and demonstrate a high level of Theileria sp. (China) infestation in Gannan Tibetan Autonomous Region. The variance in positive rates in the different geographical areas may be attributed among others to the collection time point, different herding modes, disease prevention conditions, and the age of the animals.

The evaluation of the sera collected every month from a stable herd of 44 goats over the time course of 1 year is shown in Fig. 1. The monthly average positive rate varied from 36.4 to 68.2%, with the average value of anti-Theileria antibody positivity starting to increase in March and reaching the highest level of 68.2% in May. This high level remained stable until July before starting to decrease. There was also a minor increase in antibody titres in November and February. The lowest antibody titres were observed from January to March.

Fig. 1
figure 1

Seroprevalence of anti-Theileria sp. (China) antibodies in a stable flock of 44 goats over the time course of 1 year

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Discussion

From 1958 on when ovine theileriosis was first reported in China (Yang et al. 1958), epidemiological investigations showed that the disease was widespread in Western China including Gansu, Ningxia, Inner Mongolia, Qinghai, Sichuan and Shannxi (Qiu and Yuan 1982; Yu et al. 1982). These survey data were based on microscopic examination, transmission experiments or clinical symptoms. All of these survey methods were useful but not efficient for large-scale investigation of samples. An ELISA based on whole crude Theileria sp. (China) antigen generated from piroplasms was developed by Gao et al. for diagnosis of theileriosis in 2002. The recombinant protein-based ELISA for detection of Theileria sp. (China) infection was not available for the study at this time point; however, comparative analysis showed a good correlation between the two tests (Miranda et al. 2006). Using the former detection system, the analysis of 1,169 sera samples collected from seven districts of three counties showed that the positive rates were between 27.8 and 83.3%, with an average rate of 70.1%. This result indicates that the majority of small ruminants in the Tibet area are suffering serious infection with Theileria sp. (China). Thus, the situation of ovine theileriosis is a severe problem in the Tibetan region, and both the local government and herdsmen are faced with taking more efficient measures to control the disease via chemical treatment of the animals or via avoidance of exposure to the vector tick in the disease seasons.

In the annual survey of a stable herd of 44 goats, the dynamics of positive rates showed that these increased in March and reached peak values in May, lasting until July. These results correlate with the biological activity of the vector tick of Theileria sp. (China), H. qinghaiensis, as it can be found on grass from the end of March to July, with April and May being peak months (Luo and Yin 1997; Yin et al. 2002a; Yuan et al. 2002). The increase in positive rates in November correlates with the observation of increased disease incidence in autumn (Ahmed et al. 2002), the smaller increase in February remains at this point unexplained. In this respect, it is interesting to note that experimentally infected animals exhibit a similar two-peaked annual course of antibody response (Miranda et al. 2006).

In summary, these data indicate that it is essential and necessitous to prevent infection and to treat the sheep and goats in Tibetan areas, whereby the best time for preventive measures should be at the end of March and for therapeutic measures from April until July.