328 faecal samples were screened by the method of saturated salt water floatation, and Eimeria sp. coccidia oocysts were identified in 198 samples (Fig. 1). The positive rate of infection was 60.37%, and the OPG value ranged from 200 to 98,600. The positive rates of intestinal parasites detected in the feces of forest musk deer in 7 forest musk deer farms in Lixian, Maoxian, Ma’erkang, Dujiangyan, Hanyuan and Luding and Shaanxi were quite different, with the infection rates of 91.84% (45/49) in Ma’erkang, 63.89% (23/36) in Luding, 57.69% (30/52) in Maoxian, 57.14% (31/54) in Lixian, 54.55% (30/55) in Dujiangyan, 48.78% (20/41) in Weinan and 46.34% (19/41) in Hanyuan, respectively.
The rate of intestinal Eimeria sp. positivity varies widely at different altitudes. The 7 forest musk deer farms are distributed at an altitude of 500 ~ 3000 m, of which Weinan and Dujiangyan are at an altitude of 0 ~ 1000 m, Hanyuan and Luding are at an altitude of 1000 ~ 2000 m, and Maoxian, Lixian and Ma’erkang are at an altitude of 2000 ~ 3000 m. The positive rates of Eimeria sp. were 52.08% (50/96), 54.55% (42/77) and 68.38% (106/155) at altitudes of 0 ~ 1000 m, 1000 ~ 2000 m and 2000 ~ 3000 m, respectively. The OPG values were 100 ~ 32,600, 200 ~ 57,200 and 800 ~ 98,600, respectively. The positive rate of intestinal Eimeria sp. infection (68.38%) in captive forest musk deer at 2000 ~ 3000 m above sea level was significantly higher than that at 0 ~ 1000 m (52.08%) and 1000 ~ 2000 m (54.55%) above sea level.
In this study, the positive rates of fecal Eimeria sp. detection in the spring and autumn of captive forest musk deer in 7 regions were quite different, and the intestinal Eimeria sp. infection was more serious in spring. The positive rates in spring and autumn were 66.23% (100/328) and 55.37% (98/328), respectively. The OPG values were 200 ~ 98,600 and 100 ~ 12,600, respectively.
Nested Pcr Amplification Of 18s Rrna
All Eimeria sp. positive samples were able to amplify 18S rRNA target gene fragment by nested PCR, the fragments from approximately to 830 bp (Fig. 2).
Pcr Identification Of 18s Rrna Gene Clone
The template of Eimeria sp. 18S rRNA gene clone was identified by routine PCR. The results of PCR products detected by agarose electrophoresis are as follows (Fig. 3).
Molecular Identification Results
After comparing and editing the sequencing results of Eimeria sp., the 18S rRNA gene sequence results of some samples were identical. The 132 18S rRNA gene sequences obtained by sequencing were divided into 34 species, including Maoxian (MX1-7), Dujiangyan (DJY1-5), Ma’erkang (MEK1-8), Lixian (LX1-3), Luding (LD1-2), Hanyuan (HY1-3) and Weinan (SX1-6), the GenBank accession numbers were shown in Table 3. The 18S rRNA gene sequences of 34 types of Eimeria were compared and analyzed in this experiment. The results showed that the intraspecific similarity was very high (Fig. 4). The sequence similarity of 18S rRNA genes of 34 types was 90.5% ~ 100%. The similarity of Maoxian (MX1-7) was 96.8% ~ 99.0%, Dujiangyan (DJY1-5) 95.0% ~ 96.8%, Ma’erkang (MEK1-8) 96.1% ~ 99.5%, Lixian (LX1-3) 95.2% ~ 97.0%, Luding (LD1-2) 92.6%, Hanyuan (HY1-3) 95.1% ~ 100%, and Weinan (SX1-6) 98.8%ཞ99.0%.
The 18S rRNA gene sequence of Eimeria sp. determined in this study was located in two branch systems respectively, and it was under the same branch with Eimeria alabamensis from Bos taurus and Eimeria faurei from Ovis aries, which indicated that they were closely related. DJY3 and Eimeria ahsata were in the same branch (Fig. 5). The 18S rRNA gene sequences of 34 types of Eimeria sp. were 91.0% ~ 98.8% similar to those of Eimeria cylindrica from Japan Bos taurus, the similarity with Eimeria faurei from Turkey Ovis aries was 91.3% ~ 99.3%, the similarity with Eimeria ahsata from Canada Ovis aries was 91% ~ 99.3%, and the similarity with Eimeria bovis and Eimeria alabamensis of Canada Bos taurus was 90.6% ~ 99.0% and 91.6% ~ 98.0%, respectively. The similarity with Eimeriidae auburnensis from Turkey Bos taurus was 91.0% ~ 99.6%, and the similarity with Isospora ohioensis was 80.8% ~ 87.3%.
The 18S rRNA gene sequences of 34 types of Eimeria sp. obtained in this study had base variations (Table 3). There was only one type of Eimeria sp. 18S rRNA gene sequence base variation in sequence base positions 67, 166, 203, 363, 554, 668 and 695, and the bases at the variation positions were (A-G), (A-G), (A-G), (T-C), (C-T), (T-G), and (A-G), respectively. Only the 18S rRNA gene sequence of Eimeria sp. in Weinan had no variation at base positions 57, 411, 431, 456, 507 and 583. Both Dujiangyan and Ma’erkang (DJY1, MEK2, MRK7) changed from base C to T at base positions 427 and 568. The difference between Ma’erkang and Weinan was smaller than that in other regions.
Table 3 34 nucleotide mutation sites of Eimeria sp. 18S rRNA gene sequence -Additional file 1