Identification of zoonotic Enterocytozoon bieneusi genotypes in pre-weaned Korean native calves

doi:10.21203/rs.2.22405/v1

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Identification of zoonotic Enterocytozoon bieneusi genotypes in pre-weaned Korean native calves

https://doi.org/10.21203/rs.2.22405/v1

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Background: Enterocytozoon bieneusi is the most common species of microsporidia that can infect humans and various animals worldwide. To date, information on the prevalence and genotypes of E. bieneusi infection in cattle in the Republic of Korea is limited. Therefore, in this study, we aimed to investigate the prevalence and genotypes of E. bieneusi circulating in pre-weaned Korean native calves and determine the age pattern of E. bieneusi infection and the relationship between E. bieneusi and diarrhea.

Results: The overall prevalence of E. bieneusi was 16.9% (53/314) in pre-weaned calves by PCR. The prevalence of E. bieneusi was the highest in September (36.2%), followed by March (28.3%). E. bieneusi infection (c 2 = 5.82, P = 0.016) was associated with diarrhea in calves. The present results indicated that E. bieneusi infection was statistically associated with calf age (c 2 = 11.61, P = 0.003); the prevalence of E. bieneusi was significantly higher in calves aged 21-40 days (odds ratio = 2.90, 95% confidence interval: 1.54-5.45; P = 0.001) than in those aged 1-20 days. Interestingly, E. bieneusi infection showed an association with diarrhea only in calves aged 1-20 days (c 2 = 5.82, P = 0.010). Five genotypes, BEB4 ( n = 12), BEB8 ( n = 23), CHN6 ( n = 1), I ( n = 1), and J ( n = 16), were identified, and all these genotypes belonged to Group 2. The genotype BEB8 was the most prevalent in all age groups regardless of diarrhea. On the other hand, the genotype I was identified only in one calf aged 10 days with diarrhea. Except for CHN6, the four other genotypes were mostly observed in cattle, and all of them have zoonotic potential.

Conclusions: This is the first report of the genotypes BEB4 and CHN6 in pre-weaned Korean native calves. The results revealed the presence of zoonotic E. bieneusi in pre-weaned Korean native calves, demonstrating that cattle may play an important role as a reservoir host in E. bieneusi transmission to humans.

Small Animal Medicine

Enterocytozoon bieneusi

pre-weaned Korean native calf

genotype

zoonotic infection

Microsporidia are obligate intracellular protozoan-like fungi that infect a wide range of invertebrates and vertebrates including humans [1]. Among approximately 17 human-pathogenic microsporidian species, Enterocytozoon bieneusi is the most common [2]. E. bieneusi usually causes gastrointestinal illnesses such as wasting syndrome and chronic diarrhea in immunocompromised patients (AIDS or organ transplant recipients, patients with cancer); however, it results in asymptomatic and symptomatic infections in immunocompetent individuals [3–7]. E. bieneusi is mainly transmitted by the fecal-oral route, and E. bieneusi spores from seemingly healthy animals, humans, contaminated water or food would be potential sources of infection [8]. Despite the clinical and public health importance of E. bieneusi, the implication of E. bieneusi has not been emphasized because the incidence rate of E. bieneusi infection is low in most countries.

Genotyping of E. bieneusi and assessment of its host specificity and zoonotic potential are dependent on the sequence analysis of the ribosomal internal transcribed spacer (ITS) [9]. Currently, 474 E. bieneusi genotypes have been identified in various hosts. By phylogenetic analysis, the genotypes of E. bieneusi have been clustered into at least 11 groups (groups 1−11) [10]. Group 1 contains the most genotypes found in humans and is considered to be zoonotic. Groups 2–11 have a narrow host range and are associated with specific hosts (ruminants, non-human primates, horses, and dogs) and wastewater [11, 12]. To date, more than 50 E. bieneusi genotypes have been identified in cattle, most of which belong to Group 2 [13]. Among them, some genotypes (BEB4, BEB6, I, and J) were detected in humans [14–17], suggesting that cattle can serve as potential reservoirs of human infection.

According to several studies, BEB4, I, and J are common genotypes of E. bieneusi genotypes found in pre-weaned calves worldwide [16, 18–20]. However, there is limited information available about the infection rates and genotype distribution of E. bieneusi in pre-weaned Korean native calves. In this study, we aimed to identify the prevalence and genotypes of E. bieneusi circulating in pre-weaned Korean native calves and determine the age pattern of E. bieneusi infection and the relationship between E. bieneusi and diarrhea.

Prevalence of E. bieneusi

The overall prevalence of E. bieneusi was 16.9% (53/314) in pre-weaned Korean native calves regardless of diarrhea. Among 10 different farms examined, E. bieneusi was detected in 6 farms (Table 1). We compared the infection rate of E. bieneusi according to the month. As shown in Table 2, the prevalence of E. bieneusi was the highest in September (36.2%), followed by March (28.3%) and October (14.7%); however, E. bieneusi infection was not detected in July and August. When compared E. bieneusi infection according to the fecal consistency, 11.9% and 22.1% were found in both diarrheic and normal feces, respectively. E. bieneusi infection was associated with diarrhea (χ² = 5.82, P = 0.016; Table 3). Co-infection with E. bieneusi and Cryptosporidium parvum was not detected; however, co-infection with E. bieneusi and Giardia duodenalis was observed in diarrheic (6.3%, 10/314) and normal feces (1.9%, 3/314). Although there was no statistical significance, the risk of diarrhea was increased by 3.36-fold during co-infection with E. bieneusi and G. duodenalis (95% confidence interval (CI): 0.91−12.43; P = 0.056; Table 4). E. bieneusi-positive samples were compared according to the age group of the calves. As shown in Table 5, the prevalence of E. bieneusi was the highest in calves aged 21−40 days, followed by 41−60 days and 1−20 days (χ² = 11.61, P = 0.003). The risk of being positive to E. bieneusi was 2.9-fold higher in calves aged 21−40 days (95% CI: 1.54−5.45; P = 0.001) than in those aged 1−20 days. The association between E. bieneusi infection and diarrhea according to the age group was analyzed by chi-square test. E. bieneusi infection was found to be associated with diarrhea only in calves aged 1−20 days (χ² = 6.61, P = 0.010; Table 6).

Table 1

Prevalence and genotypes of E. bieneusi in pre-weaned Korean native calves
Region	Specimens	No. of positive samples	ITS genotype (No.)
Anseong	39	4	BEB8 (n = 2), J (n = 2)
Geochang	78	22	BEB4 (n = 3), BEB8 (n = 8), J (n = 11)
Gimje	71	1	I (n = 1)
Gyeongju	6	0	−
Jeongeup	1	0	−
Mungyeong	82	21	BEB4 (n = 9), BEB8 (n = 9), CHN6 (n = 1), J (n = 2)
Naju	1	0	−
Sangju	2	1	J (n = 1)
Yechoen	1	0	−
Youngju	33	4	BEB8 (n = 4)
Total	314	53	BEB4 (n = 12), BEB8 (n = 23), CHN6 (n = 1), I (n = 1), J (n = 16)

Table 2

Prevalence of E. bieneusi in pre-weaned Korean native calves according to the month
Month	No. of examined calves	No. of positive calves	Positive rate (%)
January	2	0	0%
March	53	15	28.3%
April	68	6	8.8%
May	27	3	11.1%
June	23	1	4.3%
July	12	0	0%
August	7	0	0%
September	47	17	36.2%
October	75	11	14.7%
Total	314	53	16.9%

Table 3

Association between diarrhea and presence of E. bieneusi in pre-weaned Korean native calves
Variables	No. (%) of E. bieneusi-positive samples	No. (%) of E. bieneusi-negative samples	Total	χ² (P value)	OR (95% CI)
Fecal consistency
Diarrhea	19 (11.9%)	141 (88.1%)	160 (100)	5.82 (0.016)	0.48 (0.26–0.88)
Non-diarrhea	34 (22.1%)	120 (77.9%)	154 (100)	5.82 (0.016)	0.48 (0.26–0.88)

Table 4

Detection rates of all pathogen species in pre-weaned Korean native calves according to the diarrhea status
Pathogen	Positive in diarrhea samples (n = 160)	Positive in non-diarrhea samples (n = 154)	χ² (P value)	OR (95% CI)
Cryptosporidium parvum	11 (6.9%)	14 (9.1%)	0.53 (0.468)	0.74 (0.32–1.68)
Giardia duodenalis	23 (14.4%)	18 (11.7%)	0.50 (0.480)	1.27 (0.66–2.46)
Enterocytozoon bieneusi	19 (11.9%)	34 (22.1%)	5.82 (0.016)	0.48 (0.26–0.88)
E. bieneusi + G. duodenalis	10 (6.3%)	3 (1.9%)	3.66 (0.056)	3.36 (0.91–12.43)

Table 5

Age distribution of E. bieneusi in pre-weaned Korean native calves
Age (Days)	Frequency of E. bieneusi positivity (%)	χ² (P value)	P value	OR	95% CI
1−20 (Ref.)	19/173 (11.0%)	11.61 (0.003)	–	1.00	–
21−40	30/114 (26.3%)		0.001	2.90	1.54–5.45
41−60	4/17 (14.8%)		0.563	1.41	0.44–4.51

Table 6

Association between diarrhea and presence of E. bieneusi in pre-weaned Korean native calves according to the age

Age (Days)	Fecal consistency	Frequency of E. bieneusi positivity (%)	χ² (P value)	OR	95% CI
1−20	Diarrhea	6/102 (5.9%)	6.61 (0.010)	0.28	0.10–0.77
	Non-diarrhea	13/71 (18.3%)	6.61 (0.010)	0.28	0.10–0.77
21−40	Diarrhea	10/44 (22.7%)	0.48 (0.490)	0.74	0.31–1.76
	Non-diarrhea	20/70 (28.6%)	0.48 (0.490)	0.74	0.31–1.76
41−60	Diarrhea	3/14 (21.4%)	1.01 (0.596)	3.27	0.30–36.31
	Non-diarrhea	1/13 (7.7%)	1.01 (0.596)	3.27	0.30–36.31

Genotypes of E. bieneusi

To determine the genotypes of E. bieneusi detected in pre-weaned Korean native calves, 53 ITS-positive samples were sequenced. Five distinct genotypes, BEB4 (n = 12), BEB8 (n = 23), CHN6 (n = 1), I (n = 1), and J (n = 16), were identified. Among them, the genotype BEB8 was the most prevalent in pre-weaned calves regardless of diarrhea. On the other hand, the genotypes CHN6 and I were found only in one calf aged 16 days and 10 days with diarrhea, respectively. In particular, the BEB8 and J genotypes were detected in all age groups. BEB4 was found only up to 40 days of age and only in two farms. The diversity of the genotypes identified was decreased with age (Table 7). As shown in Table 1, the distribution of E. bieneusi genotypes on the farms was different. Three farms (Gimje, Sangju, and Youngju) had only one genotype, whereas the others had two to three genotypes (Table 1). Of the 53 ITS-positive samples, 16 sequences were included in the phylogenetic tree, and all these isolates belonged to Group 2 (Fig. 1). Except for CHN6, the four other genotypes have been mostly observed in cattle. The CHN6 identified in this study showed 98.5% homology with an isolate in feces found from human in China (Fig. 1). To the best of our knowledge, this is the first report of the genotypes BEB4 and CHN6 in pre-weaned Korean native calves.

Table 7

Genotype distribution of E. bieneusi according to the age in pre-weaned Korean native calves
	BEB4	BEB8	CHN6	I	J	Total
1−20	4	8	1	1	5	22
21−40	8	12	−	−	10	30
41−60	−	3	−	−	1	4
Total	12	23	1	1	16	53

The present study showed that the infection rate of E. bieneusi in pre-weaned Korean native calves was 16.9%, which is similar to that reported in other studies for cattle in the Republic of Korea (ROK) and several countries [16, 21–24]. The prevalence of E. bieneusi in pre-weaned calves varied from 0 to 50% depending on the farm. The differences could be attributed to nutrition, herd management practices, health of the animal, and hygiene. Interestingly, E. bieneusi infection seems to be closely related to seasonal differences. According to our results, E. bieneusi infection tended to occur mainly in September (36.2%) and March (28.3%); in contrast, the incidence rate of E. bieneusi was rather low in the warmer seasons with no infection in July and August. This result was different from that of a previous study showing a higher prevalence in warmer seasons in the ROK [21]. The differences between the two groups may be explained by the difference in the number of samples collected each month and the age of the calf. Nevertheless, our results are somewhat consistent with those a study conducted in China, which reported a higher prevalence in spring [25]. Although the results are inconclusive, the transmission of E. bieneusi may be related to seasonal variations. Further studies are required to investigate the association between E. bieneusi infection and seasonal variations.

E. bieneusi infection was related to diarrhea according to 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. Interestingly, among the three pathogens examined, the infection rate of E. bieneusi was the highest in pre-weaned Korean native calves. This may have been overlooked in diagnostic tests due to uncertainty regarding the role of E. bieneusi as a pathogen in calf diarrhea. Moreover, it is possible that the importance of E. bieneusi is not greatly recognized in fields. The present results 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. In this study, the number of positive samples co-infected with two pathogens was small; thus, these results did not demonstrate an association between diarrhea and co-infection in pre-weaned calves. Although it remains unclear, such co-infection may 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 5). 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 maternal antibodies [26]. To date, several studies have demonstrated the age-related prevalence pattern of E. bieneusi infection [18, 22, 23, 27]. However, in contrast to our findings, the prevalence of E. bieneusi has been found to increase with age [18, 28, 29]. 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 the age group. In addition, there is no information on the transmission route of E. bieneusi on 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.

The association between E. bieneusi infection and diarrhea was investigated 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 main 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 regarded 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 five known genotypes (BEB4, BEB8, CHN6, I, and J) belonging to 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 in cattle [23, 30, 31] but also in bats [32], rabbits [33], and monkeys [34], 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 was detected in only one calf. In a previous study in the ROK, the genotype I was found in three cattle. This can be attributed to the low incidence of genotype I in cattle in the ROK compared with other countries. This study was the first identification of the genotype CHN6 in pre-weaned Korean native calves. This isolate showed 98.5% homology with an isolate in human feces (MN136773) obtained in China. In addition, 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 farm management system. All genotypes identified in this study have a possible zoonotic potential, suggesting that cattle may play an important role as a reservoir host in E. bieneusi transmission to humans.

The presence and genotypes of E. bieneusi were detected in pre-weaned Korean native calves. E. bieneusi infection was associated with diarrhea in calves aged 1−20 days, and the prevalence of E. bieneusi was significantly high in calves aged 21−40 days. ITS sequencing identified five genotypes (BEB4, BEB8, CHN6, I, and J), with BEB8 as the most prevalent genotype in pre-weaned Korean native calves. The identification of zoonotic genotypes in pre-weaned calves suggests that these animals could play an important role as reservoir hosts for zoonotic infections.

Ethics statement

All procedures and possible consequences were explained to farm owners/managers of the surveyed farm. Written informed consent was obtained for the collection of fecal samples from the Korean native calves’ owners. Also, permission from farm owners/managers was obtained before collection of fecal samples.

Sample collection

From January to October 2018, a total of 314 fecal samples were collected directly by an experienced veterinarian from the rectum of pre-weaned Korean native calves (£60 days) in 10 different farms in the ROK, transported to the Animal Immunology Laboratory of Kyungpook National University in cooler with ice packs, and stored at 4°C before DNA extraction. The fecal consistency of each calf was categorized as normal or diarrheic according to its physical characteristics.

DNA extraction and PCR amplification

Genomic DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) from approximately 200 mg of each fecal sample according to the manufacturer’s instructions, and then stored at -20°C until used in PCR analysis. E. bieneusi was screened based on the ITS region of the rRNA by nested PCR under the following conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, and a final extension at 72°C for 10 min [35]. Cryptosporidium parvum and Giardia duodenalis infections were also detected using the 60-kDa glycoprotein (gp60) and β-giardin genes [36-38]. Secondary PCR products were separated by electrophoresis on 1.5% agarose gels and then visualized after staining with ethidium bromide. In this study, only samples showing a good sequencing result were considered to be positive for E. bieneusi.

Sequencing and phylogenetic analysis

The secondary PCR products were purified using the AccuPower PCR Purification Kit (Bioneer, Daejeon, ROK) and used for direct sequencing (Macrogen, Daejeon, ROK). To determine the genotype of E. bieneusi, the nucleotide sequences obtained in this study were aligned using ClustalX and compared with the reference sequences from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov). A phylogenetic tree was constructed based on nucleotide alignments using the maximum-likelihood method implemented in MEGA 7 software and bootstrap analysis was used to evaluate the robustness with 1000 replicates.

Statistical analysis

Statistical analysis was performed using SPSS Statistics 25 software package for Windows (SPSS Inc., Chicago, IL, USA). Chi-square (c²) was used to compare the associations between diarrhea and infection rate of E. bieneusi in each age range or all ages investigated in this study. In addition, the prevalence of E. bieneusi for each age range was determined using binary univariate logistic regression models. The odds ratio (OR) and 95% CI were calculated to assess the likelihood of association. A P value of £0.05 was considered to be statistically significant.

E. bieneusi

Enterocytozoon bieneusi; CI:confidence interval; C. parvum; Cryptosporidium parvum; G. duodenalis:Giardia duodenalis; ITS:internal transcribed spacer; OR:odds ratio; ROK:Republic of Korea

Ethics approval and consent to participate

This study did not receive approval from the Institutional Animal Care and Use Committee (IACUC) at Kyungpook National University, because the IACUC at this University evaluates laboratory animals maintained within indoor facilities and not at outdoor animals. A local veterinarian took all fecal samples and sent them to us.

Consent for publication

Not applicable

Availability of data and material

All data generated or analyzed during this study are included in the article.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by the National Research Foundation of Korea (NRF), funded by the Korea government (MSIP) (No. 2018R1D1A1B07048271).

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author’s contributions

KSC designed the research and wrote the manuscript. SWH, SUS, and JHR performed the experiments. SHK analyzed the data. All authors read and approved the final manuscript.

Acknowledgements

We thank Jeong-Byoung Chae, DVM for collecting feces and Yoon-Joo Lee for helping with DNA preparation.

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Identification of zoonotic Enterocytozoon bieneusi genotypes in pre-weaned Korean native calves

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