The present study showed that the infection rate of E. bieneusi in pre-weaned Korean native calves was 16.9%, which was similar to that reported in other studies for cattle in the ROK and several other countries [17, 26-29]. The prevalence of E. bieneusi in pre-weaned calves varied from 0% to 50% depending on the farm. This variation could be attributed to nutrition, herd management practices, health of the animal, and hygiene. Interestingly, E. bieneusi infection appears to be closely related to seasonal differences. According to our results, E. bieneusi infection tended to occur primarily in September (36.2%) and March (28.3%); in contrast, the incidence rate of E. bieneusi was rather low in warmer seasons with no infections in July and August. This result was different from that of a previous study that had reported a higher prevalence during warmer seasons in the ROK . The differences between these two studies might be explained by the difference in the number of samples collected each month and the age of the calf. However, our results were fairly consistent with those of a study conducted in China, which reported a higher prevalence in spring . Although the results are inconclusive, the transmission of E. bieneusi may be related to seasonal variations. Thus, further studies are required to investigate the association between E. bieneusi infection and seasonal variations.
In this study, E. bieneusi infection was associated with diarrhea according to the chi-square analysis; however, the infection rate was not high in diarrheic feces. Although E. bieneusi was detected in diarrheic feces, it is unlikely that E. bieneusi is associated with diarrhea in pre-weaned calves (OR=0.48, 95% CI: 0.29-0.88; P = 0.016). However, a previous study performed in China revealed that E. bieneusi infection was increased 2.5-fold in pre-weaned calves with diarrhea (95% CI: 1.7-3.8; P = 0.000) compared with those without diarrhea and most of all, E. bieneusi infection was associated with diarrhea . The difference between the two groups could be explained by the number of samples and the age of the calf. Thus, the relationship between E. bieneusi infection and diarrhea should be determined through further investigation.
Interestingly, among the three pathogens examined, the infection rate of E. bieneusi was the highest in pre-weaned Korean native calves. This might have been overlooked in diagnostic tests due to uncertainty regarding the role of E. bieneusi as a pathogen in calf diarrhea. Furthermore, it is possible that the significance of E. bieneusi has not been prominently recognized in the fields. Results of the present study showed that co-infection with E. bieneusi and G. duodenalis was not statistically significant (P = 0.056); however, it was 3.36-fold more likely to cause diarrhea (95% CI: 0.91-12.43) compared with E. bieneusi single infection in calves. Our result was inconsistent with that reported in China; co-infection with E. bieneusi and G. duodenalis was significantly associated with diarrhea . In this study, the number of positive samples co-infected with two pathogens was small; thus, these results failed to demonstrate an association between diarrhea and co-infection in pre-weaned calves. Although it remains unclear, such co-infection might increase the severity and duration of diarrhea in calves. More epidemiological investigations are required to determine whether the occurrence of diarrhea is more common in calves co-infected with E. bieneusi and G. duodenalis.
The prevalence of E. bieneusi in calves was significantly associated with the age of the calf (P = 0.003). The infection rate of E. bieneusi was the highest in calves aged 21-40 days, followed by calves aged 41-60 days, and 1-20 days. In comparison with calves aged £ 20 days, the risk of E. bieneusi infection was 2.9-fold higher in calves aged 21-40 days (P = 0.001; Table 3). A possible explanation is that the immune status of calves in this age group may be more susceptible to E. bieneusi infection due to the loss of the maternal antibodies . To date, several studies have demonstrated the age-related prevalence pattern of E. bieneusi infection [19, 27, 28, 33]. However, in contrast to our findings, the prevalence of E. bieneusi has been found to increase with age [19, 34, 35]. In the ROK, there are limited studies on E. bieneusi infection in cattle; thus, it is not possible to compare the prevalence of E. bieneusi according to age group. In addition, there is no information on the transmission route of E. bieneusi on the farms examined; however, E. bieneusi infection may be related to the hygiene status of farms rather than the calf age. Therefore, to prevent E. bieneusi infection, the farming management system should be improved, which could include no contact with contaminated food and water, cleaning, and disinfection.
We also investigated the association between E. bieneusi infection and diarrhea according to the age group. Our results revealed that there was a significant correlation between E. bieneusi infection and diarrhea in calves aged 1-20 days (P = 0.010). However, this has been shown to be associated with a low incidence of diarrhea in E. bieneusi-infected calves. Based on the results, it is unlikely that E. bieneusi is the primary pathogen that causes diarrhea in pre-weaned Korean native calves. Cattle may be a source of environmental contamination by E. bieneusi. Therefore, E. bieneusi infection in calves should be considered as a zoonotic potential rather than a causative agent of diarrhea.
In the present study, sequence analysis of the ITS gene from 53 E. bieneusi-positive isolates identified six genotypes (BEB4, BEB8, J, BEB8-like, KCALF1, and KCALF2) belonging to zoonotic Group 2. Unlike previous studies, the genotype BEB8 was the most prevalent in pre-weaned Korean native calves and was found in 45.3% (24/53) of the positive samples. Moreover, this genotype was commonly identified in all age groups regardless of diarrhea. Several studies have reported that the genotype BEB8 can be found in not only cattle [28, 36, 37] but also bats  and rabbits , indicating that this genotype might have a potential risk for zoonotic infection in humans. The genotype J, identified in 30.2% (16/53) of E. bieneusi-positive samples, was the second most common genotype in all age groups. BEB4 was the third most common genotype and found in calves only up to 40 days. BEB4 has been identified as a zoonotic genotype in cattle in many countries; however, it was first detected in the ROK. Interestingly, the genotype I with a wide range of hosts was not detected in this study. In contrast, a previous study conducted in the ROK reported the presence of the genotype I in three cattle . This can be attributed to the low incidence of genotype I in cattle in the ROK compared to that of other countries. The present study reported the identification of three novel genotypes in pre-weaned Korean native calves, indicating that high genetic diversity exists in the E. bieneusi ITS region. Furthermore, the genotypes BEB4, BEB8, and J were common genotypes in pre-weaned Korean native calves. The differences in the distribution of E. bieneusi in pre-weaned calves according to farms may be attributed to the geographical location and the farm management system. All genotypes identified in this study have a possible zoonotic potential, suggesting that cattle play an important role as a reservoir host in E. bieneusi transmission to humans.