Abstract:

Objective To clarify the prevalence and main genotypes of Enterocytozoon bieneusi infection in dairy cows in Yinchuan area of Ningxia Province. Methods From August 2021 to August 2022, cow fresh fecal samples (including diarrheal and non-diarrheal samples) were collected from 6 scaled dairy farms in Yinchuan City and the surrounding counties. The genomic DNA was extracted from the fecal samples, and nested PCR was performed using E. bieneusi internal transcriptional spacer (ITS) primers. The PCR amplicaons were seqeuced, and sequence alignment was conducted using BLAST to identify the genotype of E. bieneusi. SPSS 25.0 software was used for statistical analysis, and χ² test for comparing the differences in positive rates between the cows of different months of age. Results A total of 772 fresh cow fecal samples were collected. The E. bieneusi positive rate from nested PCR amplification was 8.7% (67/772). The target band size was approximately 400 bp. The E. bieneusi positive rate found in different farms ranged from 4.8% (6/124) to 11.5% (18/156), with statistically significant differences (χ² = 30.828, P < 0.05). The highest positive rate was found in cows aged 5-7 months (15.6%, 23/147) and the lowest was found in cows aged above 12 months (3.4%, 5/149). There was a statistically significant difference in the positive rate of E. bieneusi among cows of different months of age (χ² = 59.529, P < 0.05). The E. bieneusi positive rate in diarrheal samples was 10.6% (42/398), which was higher than that in non-diarrheal samples (6.7%, 25/374) (χ² = 3.419, P < 0.05). The 66 E. bieneusi sequences could be classified into 4 genotypes, including CHN4, BEB4, J and I. The accession obtained by GenBank were OR352930, OR352929, OR352932 and OR352931 respectively. 3 sequences were CHN4 (account for 4.5%), which showed a 98.4% identity to the sequence of CHN4 (HM992511) in G1. 18 sequences were BEB4 (account for 27.3%), which showed a 96.3% identity to the sequence of BEB4 (AY331008) in G2. 22 sequences were J (account for 33.3%), which showed a 93.1% identity to the sequence of J (AF135837) in G2. 23 sequences were I (account for 34.9%), which showed a 91.1% identity to the sequence of I (AF135836) in G2. The dominant E. bieneusi genotypes in cows varied with age, but BEB4, J and I presented in all age groups. The BEB4, J and I accounted for 88.1% (37/42) and 60.0% (15/25) of the total positive diarrheal samples and positive normal samples respectively, with statistically significant differences (χ² = 3.419, P < 0.05). Conclusion E. bieneusi infection was found in dariy cows in Yinchuan area of Ningxia Province, with the main genotypes BEB4, J and I.

Key words: Enterocytozoon bieneusi, Infection, Internal transcribed spacer, Nested PCR, Yinchuan