目的 分析海南省五指山猪感染的隐孢子虫虫种和毕氏肠微孢子虫基因型，掌握两种原虫的感染情况。方法 2024年6月—2025年2月，从海南省遵谭镇、琼中黎族苗族自治县、五指山市、澄迈县的4个养殖场（分别标记为A、B、C、D场）采集五指山猪新鲜粪样。提取粪样DNA，巢式PCR扩增隐孢子虫18S核糖体RNA（18S rRNA）和毕氏肠微孢子虫内转录间隔区（ITS）序列，阳性扩增产物测序后进行同源性比对，鉴定隐孢子虫虫种或毕氏肠微孢子虫基因型。采用SPSS 26.0软件进行统计学分析，阳性率差异的比较采用皮尔逊卡方（χ²）检验，并计算风险系数（OR）和95%置信区间（CI）。采用MEGA 11软件邻接法构建系统进化树。结果 共采集137份五指山猪粪样，其中有41份扩增出830 bp的隐孢子虫18S rRNA序列，69份扩增出390 bp的毕氏肠微孢子虫ITS序列。4个养殖场的五指山猪隐孢子虫总阳性率为29.9%（41/137），其中C场最高（64.3%，18/28）、B场最低（20.3%，14/69）。断奶后仔猪的隐孢子虫阳性率为57.1%（12/21），高于断奶前仔猪的7.7%（2/26）（χ² = 13.583，P < 0.05）；断奶后仔猪和成年猪的感染OR是断奶前仔猪的16.00倍（95% CI = 2.98~85.99）和5.14倍（95% CI = 1.15~23.31）。毕氏肠微孢子虫总阳性率为50.4%（69/137），其中C场最高（60.7%，17/28）、A场最低（37.5%，9/24）。断奶后仔猪的毕氏肠微孢子虫阳性率为80.9%（17/21），高于断奶前仔猪的42.3%（11/26）和成年猪的45.5%（41/90）（χ² = 7.204、8.551，均P < 0.01）；断奶后仔猪和成年猪的感染OR是断奶前仔猪的5.80倍（95% CI = 1.52~22.10）和1.14倍（95% CI = 0.47~2.76）。放牧五指山猪的毕氏肠微孢子虫阳性率为62.8%（44/70），高于舍饲五指山猪的37.3%（25/67）（χ² = 8.935，P < 0.01）；放牧五指山猪的感染OR是舍饲五指山猪的2.84倍（95% CI = 1.42~5.69）。序列比对结果显示，41条隐孢子虫18S rRNA序列均鉴定为猪隐孢子虫，与种猪隐孢子虫（C. scrofarum，GenBank登录号：PQ856498）的序列一致性达93.36%~99.74%；在系统进化树中与云南猪源种猪隐孢子虫、湖北猪源种猪隐孢子虫（GenBank登录号：PQ856498、ON149807）处于同一大分支。69条毕氏肠微孢子虫ITS序列鉴定为H、D、和EpbC 3种基因型，其中3条H基因型序列与德国猪源H基因型（GenBank登录号：AF135835.1）的一致性达98.00%，25条D基因型序列与澳大利亚猫源D基因型（GenBank登录号：MK696083.1）的一致性达97.51%~99.72%，41条EpbC基因型序列与中国熊猫源EpbC基因型（GenBank：登录号KY950535.1）的一致性达98.59%~99.73%；系统进化树中，D基因型与阿根廷人源D基因型和澳大利亚猫源D基因型（GenBank登录号：OP650902、MK696083）处于同一分支，EbpC基因型与中国熊猫源EpbC基因型（GenBank登录号：KY950535.1）处于同一分支，H基因型与德国猪源H基因型（GenBank登录号：AF135835.1）处于同一分支。种系发育分析显示3个基因型均属于1组亚群。结论 海南五指山猪存在毕氏肠微孢子虫和隐孢子虫感染，隐孢子虫感染虫种为种猪隐孢子虫，毕氏肠微孢子虫主要基因型为H、D、EbpC。

Objective To analyze the species of Cryptosporidium and the genotype of Enterocytozoon bieneusi in Wuzhishan pigs in Hainan, and to master the infection of the two protozoa. Methods From June 2024 to February 2025, fresh fecal samples of Wuzhishan pigs were collected from 4 farms in Zuntan Town, Qiongzhong Li and Miao Autonomous County, Wuzhishan City and Chengmai County of Hainan Province (marked as A, B, C and D farms, respectively). DNA was extracted from fecal samples, and the 18S ribosomal RNA (18S rRNA) of Cryptosporidium and the internal transcribed spacer (ITS) sequence of E. bieneusi were amplified by nested PCR. The positive amplification products were sequenced and compared to identify Cryptosporidium species or E. bieneusi genotypes. SPSS 26.0 software was used for statistical analysis. Pearson chi-square (χ²) test was used to compare the differences in positive rates, and the risk coefficient (OR) and 95% confidence interval (CI) were calculated. The phylogenetic tree was constructed by MEGA 11 software adjacency method. Results A total of 137 Wuzhishan pig manure samples were collected, of which 41 were amplified with 830 bp of Cryptosporidium 18S rRNA sequence and 69 were amplified with 390 bp of E. bieneusi ITS sequence. The total positive rate of Cryptosporidium in Wuzhishan pigs from four farms was 29.9% (41/137), with the highest in C farm (64.3%, 18/28) and the lowest in B farm (20.3%, 14/69). The positive rate of Cryptosporidium in post-weaning piglets was 57.1% (12/21), which was higher than 7.7% (2/26) in pre-weaning piglets (χ² = 13.583, P < 0.05). The OR of post-weaning piglets and adult pigs was 16.00 times (95% CI = 2.98-85.99) and 5.14 times (95% CI = 1.15-23.31) higher than that of pre-weaning piglets. The total positive rate of E. bieneusi was 50.4% (69/137), with the highest in C farm (60.7%, 17/28) and the lowest in A farm (37.5%, 9/24). The positive rate of E. bieneusi in post-weaning piglets was 80.9% (17/21), which was higher than that in pre-weaning piglets (42.3%, 11/26) and adult pigs (45.5%, 41/90) (χ² = 7.204, 8.551; both P < 0.01). The OR of post-weaning piglets and adult pigs was 5.80 times (95% CI = 1.52-22.10) and 1.14 times (95% CI = 0.47-2.76) higher than that of pre-weaning piglets. The positive rate of E. bieneusi in grazing Wuzhishan pigs was 62.8% (44/70), which was higher than that in barn feeding Wuzhishan pigs (37.3%, 25/67) (χ² = 8.935, P < 0.01). The OR of infection in grazing Wuzhishan pigs was 2.84 times (95% CI = 1.42-5.69) higher than that in feeding Wuzhishan pigs. The results of sequence alignment showed that 41 Cryptosporidium 18S rRNA sequences were identified as C. scrofarum, and the sequence identity with C. scrofarum (GenBank accession number: PQ856498) was 93.36%-99.74%. In the phylogenetic tree, it was in the same large branch with C. scrofarum from Yunnan pig and Hubei pig (GenBank accession number: PQ856498, ON149807). 69 ITS sequences of E. bieneusi were identified as H, D, and EpbC genotypes. Among them, 3 H genotype sequences were 98.00% identical to the H genotype of German pig (GenBank accession number: AF135835.1). The identity of 25 D genotype sequences with D genotype (GenBank accession number: MK696083.1) from Australian cats was 97.51%-99.72%, and the identity of 41 EpbC genotype sequences with EpbC genotype (GenBank accession number: KY950535.1) from Chinese pandas was 98.59%-99.73%. In the phylogenetic tree, D genotype was in the same branch with Argentinean D genotype and Australian cat D genotype (GenBank accession number: OP650902, MK696083), EbpC genotype was in the same branch with Chinese panda EpbC genotype (GenBank accession number: KY950535.1), and H genotype was in the same branch with German pig H genotype (GenBank accession number: AF135835.1). Phylogenetic analysis showed that the three genotypes belonged to one subgroup. Conclusion There were E. bieneusi and Cryptosporidium infection in Wuzhishan pigs in Hainan. The Cryptosporidium infection species was C. scrofarum. The main genotypes of E. bieneusi were H, D and EbpC.