Infection and genotype identification of Enterocytozoon bieneusi and Cryptosporidium in Wuzhishan pigs of Hainan

doi:10.12140/j.issn.1000-7423.2025.05.011

Abstract

Abstract:

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.

Key words: Enterocytozoon bieneusi, Cryptosporidium, Wuzhishan pig, Zoonotic diseases, Genotype

CLC Number:

R382.3

DONG Yatong, ZHANG Yan, LIU Guangliang, CAO Zongxi, YU Miao, CHEN Linlin, LIU Hailong, CHEN Suzhen. Infection and genotype identification of Enterocytozoon bieneusi and Cryptosporidium in Wuzhishan pigs of Hainan[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(5): 670-677.

Figures/Tables 5

Table 1

Fig. 1

Table 2

Fig. 2

Fig. 3

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寄生虫种 Parasite species	引物名称 Primer name	引物序列（5′→3′） Primer sequence (5′→3′)	退火温度/℃ Annealing temperature/℃	片段大小/bp Fragment size/bp
隐孢子虫	18S rRNA-F1	TTCTAGAGCTAATACATGCG	56	1 325
Cryptosporidium	18S rRNA-R1	CCCATTTCCTTCGAACAGGA
	18S rRNA-F2	GGAAGGGTTGTATTTATTAGATAAAG	58	830
	18S rRNA-R2	CTCATAAGGTGCTGAAGGAGTA
毕氏肠微孢子虫	ITS-F1	GATGGTCATAGGGATGAAGAGCTT	57	410
E. bieneusi	ITS-R1	TATGCTTAAGTCCAGGGAG
	ITS-F2	AGGGATGAAGAGCTTCGGCTCTG	55	390
	ITS-R2	AGTGATCCTGTATTAGGGATATT

特征 Feature	粪样份数 No. fecal sample	隐孢子虫 Cryptosporidium		毕氏肠微孢子虫 E. bieneusi
特征 Feature	粪样份数 No. fecal sample	阳性份数 No. positive	阳性率/% Positive rate/%	阳性份数 No. positive	阳性率/% Positive rate/%
粪样来源 Source of fecal samples
A场 Farm A	24	5	20.8（5/24）	9	37.5（9/24）
B场 Farm B	69	14	20.3（14/69）	35	50.7（35/69）
C场 Farm C	28	18	64.3（18/28）	17	60.7（17/28）
D场 Farm D	16	4	4/16	8	8/16
生长阶段 Growth stage
断奶前仔猪（≤ 60 d） Pre-weaned piglets (≤ 60 d)	26	2	7.7（2/26）	11	42.3（11/26）
断奶后仔猪（> 60~180 d） Post-weaned piglets (60-180 d)	21	12	57.1（12/21）	17	80.9（17/21）
成年猪（> 180 d） Adult pigs (> 180 d)	90	27	30.0（27/90）	41	45.5（41/90）
饲养方式 Feeding methods
舍饲 House feeding	67	17	25.4（17/67）	25	37.3（25/67）
放牧 Grazing	70	24	34.3（24/70）	44	62.8（44/70）
合计 Total	137	41	29.9（41/137）	69	50.4（69/137）