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All issues Volume 31 (2024) Parasite, 31 (2024) 59 Full HTML
Open Access
Issue
Parasite
Volume 31, 2024
Article Number 59
Number of page(s) 12
DOI https://doi.org/10.1051/parasite/2024061
Published online 27 September 2024

© F. Shan et al., published by EDP Sciences, 2024

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Microsporidiosis is an emerging opportunistic disease caused by microsporidia infection, affecting both invertebrates and vertebrates, including humans [20, 38]. Microsporidia constitute a vast group of parasitic eukaryotes that live inside host cells, with over 1500 species distributed among 200 different genera, of which 17 species in 10 genera have been identified as agents of human infections [28, 38]. The predominant culprit of human microsporidiosis is Enterocytozoon bieneusi, which accounts for over 90% of documented cases [20, 28, 38]. Besides humans, E. bieneusi is widely present in mammalian and avian hosts worldwide, raising concerns about the role of animal hosts in the spread of the pathogen [37]. The most likely routes of E. bieneusi transmission to humans or animals are transmission through contact with infected hosts and ingestion of spores of environmental origin [43]. Indeed, in addition to direct infection of humans, infected hosts can release E. bieneusi spores into the environment, causing contamination of agricultural products and water sources. Consequently, there have been cases of foodborne and waterborne transmission of E. bieneusi [5, 7, 11, 46].

Due to their tiny size of about 1 μm, the spores of E. bieneusi can be mistaken for food particles, fungi, bacteria, and other microsporidia, posing a challenge for accurate diagnosis using conventional microscopy techniques. The ability to correctly identify E. bieneusi relies heavily on the skills of the examiner [37, 50]. While immunodetection with monoclonal antibodies is convenient for broad epidemiological studies, it does not provide genotyping information [1, 55]. However, the advent of molecular technologies, such as polymerase chain reaction (PCR) and internal transcribed spacer (ITS) DNA sequencing of ribosomes, has allowed for the global characterization of the molecular signature of E. bieneusi in humans, various domestic and wild animals, and aquatic environments [20, 23, 37]. As of now, over 500 E. bieneusi genotypes have been identified worldwide [20]. These genotypes have been classified into 11 groups (groups 1–11) by phylogenetic analysis [20, 23]. Genotypes in groups 1 and 2 have been found in a wide range of hosts, including humans, and are likely responsible for most zoonotic or cross-species E. bieneusi infections, whereas host-adapted genotypes appear to be more common in groups 3–11 [20, 22, 23]. Conducting molecular epidemiological studies is essential for genotyping E. bieneusi strains extracted from underrepresented animal hosts to gain a better understanding E. bieneusi epidemiology and assess the role of animals in its transmission to humans.

Rodents and shrews are widely distributed throughout the world, often gathering in dense concentrations, and come into close contact with humans and domestic animals [2, 17]. This proximity poses a potential threat to public health as they can destroy and contaminate food and transmit various pathogens, particularly in agricultural settings where food is abundant [30, 33]. Research has shown that there are about 60 different genotypes of E. bieneusi in rodents, with 18 of them being capable of zoonotic transmission, affirming the part that rodents play in spreading E. bieneusi [40, 53]. Pigs are highly susceptible to E. bieneusi infection and primarily harbor group 1 genotypes; thus, they are primary hosts for pathogenic strains that affect humans. Among these, the zoonotic genotypes EbpC, EbpA, O, H, and D are the most commonly found [23, 41]. In swine farm environments, there is a significant presence of wild small mammals, including rodents and shrews, which share the same living area and can come into close contact with humans and domestic pigs [2]. Therefore, it is essential to identify the epidemiological characteristics of E. bieneusi in these animals inhabiting swine farm environments and their role in transmission to prevent its spread among humans and other animals. Our research aimed to determine the molecular prevalence of E. bieneusi in rodents and shrews within swine farm environments in China by using nested PCR and sequence analysis of the ribosomal ITS region. Furthermore, the zoonotic potential of the isolates and possible routes of transmission of E. bieneusi were assessed through genotypic identification and phylogenetic analysis.

Materials and methods

Ethical standards

The research procedure was approved by the Research Ethics Committee of Henan Agricultural University in accordance with the Chinese Laboratory Animal Administration Act of 1998. The study team obtained permission from the farm owners or managers to access the farms and collect samples.

Specimen collection

Wild small mammal trapping was conducted between March 2021 and June 2023 on 34 pig farms situated in 18 cities across Henan, Shaanxi, and Shanxi Provinces in China (Fig. 1). Trapping cages were used for capture and were deployed on pig farms 1 h before dusk and collected within 1 h after sunrise. The trapping activities were performed for 3–5 days on each farm. In total, 227 wild small mammals (136 Rattus tanezumi, 58 Rattus norvegicus, 25 Mus musculus, one Apodemus agrarius, and seven Crocidura shantungensis) were captured (Table 1), with the number of captures ranging from 1 to 23 per farm. After capture, the wild small mammals were humanely euthanized via carbon dioxide inhalation. Each animal was then individually placed in labeled bags containing information that included sex, body weight, sampling date, sampling location, farm size, farm type, and duration of pig rearing. Within 48 h, the specimens were transported in containers with ice packs to the laboratory for necropsy, which was conducted in a biosafety cabinet. Fecal and liver samples were collected from each specimen and stored at −80 °C for subsequent analysis.

thumbnail Figure 1

Map of the sampling areas and the geographical distribution of Enterocytozoon bieneusi detected in the present study. The sampled pig farms are represented by circles on the map and the numbers next to the circles represent farm numbers. The numbers of E. bieneusi genotypes on the surveyed pig farms are indicated by colored circles of different sizes.

Table 1

Prevalence and genotypes of Enterocytozoon bieneusi among rodents and shrews inhabiting pig farms in China.

DNA extraction

A QIAamp PowerFecal Pro DNA kit (QIAGEN, Hilden, Germany) was used to extract the genomic DNA from each fecal sample (approximately 200 mg), according to the manufacturer’s instructions. The extracted DNA was stored at −20 °C for subsequent PCR analysis.

Molecular analysis

Samples were screened for the presence of E. bieneusi by amplifying a 389-bp nucleotide fragment of the ITS segment using a previously described nested PCR method [39]. The negative controls for the experiment included double-distilled water, while the positive controls consisted of known DNA from cows with genotype D. Following amplification, the PCR products were separated by electrophoresis on 1% agarose gels containing DNAGREEN dye (Tiandz, Inc., Beijing, China). Positive samples were subjected to bidirectional sequencing at SinoGenoMax Biotechnology in Beijing, China. The obtained raw sequences in this study were then assembled and corrected using DNASTAR 7.1.0 (http://www.dnastar.com/) and aligned with reference sequences downloaded from GenBank. Genotypes were labeled according to the established nomenclature of the E. bieneusi 243-bp ITS region. A neighbor-joining phylogenetic tree was constructed using Mega 11 software with the Kimura-2 parametric algorithm and 1000 replicates to reveal the evolutionary relationships and zoonotic potential among the genotypes of E. bieneusi isolates.

Statistical analysis

IBM SPSS version 27.0 (IBM Corp., Armonk, NY, USA) was used to conduct the statistical analysis. The risk factors associated with E. bieneusi infections were evaluated by calculating the odds ratio and 95% confidence interval (CI) through either univariate analyses (Chi-squared test or Fisher’s exact test) and multivariate analyses (Binary logistic regression analyses). The variables with p < 0.20 in the univariate analysis were introduced in the multivariate analyses. Statistical significance was determined at a p-value threshold of <0.05.

Nucleotide sequence accession numbers

The unique nucleotide sequences obtained were submitted to the GenBank database under accession numbers PP158567–PP158577.

Results

Prevalence of Enterocytozoon bieneusi

In 39 out of 227 fecal samples, Enterocytozoon bieneusi was found, giving a prevalence rate of 17.2% (95% CI, 12.2–22.1). Among the infected individuals, there were 32 R. tanezumi (23.5%, 16.3–30.7), five R. norvegicus (8.6%, 1.2–16.1), and two M. musculus (8.0%, −3.4–19.4) (Table 1). A statistically significant difference was observed between these groups (χ2 = 8.029, p = 0.018) (Table 2). However, all individuals of A. agrarius and C. shantungensis tested negative for E. bieneusi. Regarding regional distribution, a statistically significant difference was found in the prevalence of E. bieneusi infection among the three provinces included in this study (χ2 = 16.795, p = 0.002). The prevalence rates of E. bieneusi infection in Shanxi, Shaanxi, and Henan were 50.0% (2/4), 35.3% (12/34), and 13.2% (25/189), respectively (Table 2). To ensure statistical accuracy, data from Shanxi Province were excluded from the analysis due to its small sample size. A re-analysis of infection rates between Henan and Shaanxi Provinces was then conducted, revealing a statistically significant difference in prevalence between the two provinces (χ2 = 10.139, p = 0.001). Additionally, E. bieneusi was detected in 13 of 34 pig farms (Fig. 1), with a farm-level positivity rate of 38.2% (95% CI, 21.0–55.4).

Table 2

Risk factors associated with the prevalence of Enterocytozoon bieneusi among rodents and shrews inhabiting pig farms.

Risk factors of Enterocytozoon bieneusi infection

Univariate analysis demonstrated a connection between E. bieneusi infection and region and host species (Table 2). However, factors such as host age, host sex, season, farm type, farm size, and duration of pig rearing did not influence E. bieneusi infection. Further analysis using the variables (farm size, region, and host species) included in the multivariate model identified regional factors (p = 0.006) significantly associated with E. bieneusi infection, which indicates an increased risk of rodents with E. bieneusi infection on pig farms in Shaanxi (OR = 3.46, 95% CI: 0.46–26.30) and Shanxi (OR = 4.01, 95% CI: 0.75–21.62) provinces relative to the Henan region (Table 2).

Characterization and distribution of the Enterocytozoon bieneusi genotypes

Eight distinct genotypes of E. bieneusi were identified from the ITS sequencing of 39 isolates. These comprised four known genotypes (D, EbpC, PigEBITS7, and EbpA) and four newly named genotypes (CHPR1, CHPR2, CHPR3, and CHPR4). The most prevalent genotype was PigEBITS7 (23.1%, 9/39), followed by D and EbpC (20.5%, 8/39). CHPR1 accounted for 17.9% (6/39) of the isolates, while EbpA, CHPR3, and CHPR5 each represented 5.1% (2/39). CHPR2 exhibited the lowest prevalence at 2.6% (1/39) (Table 1 and Fig. 2A). Interestingly, although the PigEBITS7 genotype was predominant, the EbpC genotype had the broadest distribution and was present on seven pig farms that tested positive for E. bieneusi (Fig. 1 and Fig. 2B).

thumbnail Figure 2

Prevalence rates and frequency of Enterocytozoon bieneusi genotypes in rodents and shrews on pig farms. (A) Prevalence rates of E. bieneusi genotypes in this study. (B) Frequency of E. bieneusi genotypes on pig farms.

Distinct patterns of E. bieneusi genotype distributions were observed among different rodent species (Table 1). In all, three rodent species that tested positive for E. bieneusi, Genotype EbpC were detected. Genotype D was detected in both R. tanezumi and R. norvegicus. Genotypes EbpA, PigEBITS7, and the newly identified genotypes (CHPR1–CHPR4) were exclusively observed in R. tanezumi.

Nucleotide sequence analysis revealed that the new genotypes CHPR1, CHPR2, and CHPR3 exhibited base substitutions in the E. bieneusi ITS region (two, at positions 81 (C → T) and 95 (G → T); two, at positions 12 (G → A) and 196 (A → G); and one, at position 19 (G → A), respectively) in comparison to the EbpC genotype from a wild boar sample (GenBank accession number MK681466). CHPR4 displayed a single base substitution at position 118 (G → A) in the E. bieneusi ITS region, aligning with genotype H (GenBank accession number AF135835) from a pig sample source (Table 3).

Table 3

Nucleotide variations in the ITS gene region of newly identified Enterocytozoon bieneusi isolates in this study.

Phylogenetic analysis of Enterocytozoon bieneusi

A phylogenetic analysis of the E. bieneusi ITS region sequences revealed that all the genotypes obtained in this study were classified under group 1 (Fig. 3). The known genotypes PigEBITS7 and D were clustered in branch 1a; the new genotypes CHPR2 and CHPR3, and the known zoonotic genotype EbpC were clustered in branch 1d; and the new genotypes CHPR1 and CHPR4 and the known zoonotic genotype EbpA were clustered in branch 1e (Fig. 3).

thumbnail Figure 3

Phylogenetic relationships of Enterocytozoon bieneusi genotype isolates in this study. Based on sequence analysis of the ITS region, the neighbor-joining method and the Kimura-2 parameter model were used to analyze relationships. Known and novel genotypes are indicated by filled squares and triangles, respectively. Bootstrap values (>50) are indicated at the nodes.

Discussion

Enterocytozoon bieneusi is a globally distributed opportunistic zoonotic pathogen [21]. Among the potential hosts of this pathogen, rodents play a significant role due to their frequent interaction with humans and domestic animals, as well as their wide presence in various environments [2, 33]. Despite the substantial research conducted on E. bieneusi infection in humans and domestic animals [35], there is limited information available regarding its prevalence in rodents, particularly in wild rodents inhabiting farm environments. Previous reports on E. bieneusi in rodents in farm settings have been scarce, with only one report each from a pig farm, cattle farm, sheep farm, and granary in Heilongjiang Province, as well as a cattle farm in Henan Province [54, 56]. Hence, this study provides the first report of E. bieneusi in rodents from pig farm environments in Henan, Shaanxi, and Shanxi Provinces of China.

Overall, 27 studies have reported the existence of E. bieneusi in rodents across eight different nations, with prevalence rates ranging from 1.1% to 100.0% (Table 4). It is noteworthy that, barring the cases of China and the USA, each country only underwent a single study. Therefore, these reported prevalence rates are likely to be theoretical rather than actual, emphasizing the need for additional comprehensive surveillance studies to ascertain the accuracy of these findings. Our research revealed an E. bieneusi infection prevalence of 17.2% (39/227), which was slightly higher than the worldwide prevalence of E. bieneusi infection in rodents (13.6%) [40]. However, this rate was significantly higher than the previously reported infection rate of 7.4% (19/242) for brown rats on a pig farm, cattle farm, sheep farm, and granary in Heilongjiang Province [56], as well as an infection rate of 4.0% (8/199) for brown rats and house mice on a cattle farm in Henan Province [54]. These differences in prevalence may be attributed to factors such as the environmental hygiene of the farms where the rodents were captured and the susceptibility of different rodent species to E. bieneusi [59]. Notably, pigs on farms with a high prevalence of E. bieneusi carriage are considered to be the main hosts of E. bieneusi [20, 23, 41], while rodents that forage freely on farms may acquire the infection through fecal contamination in intensive pig housing. The high field positivity rate (38.2%, 13/34) in our study appears to support this hypothesis. It is necessary to expand the survey and adopt a One Health approach to further investigate this discrepancy and determine the possible routes of transmission between farmed pigs and sympatric rodents.

Table 4

Prevalence and genotype distribution of Enterocytozoon bieneusi in rodents in different countries or areas.

To date, E. bieneusi infections have been reported in approximately 38 rodent species (Table 4). In this study, we investigated a total of five rodent and shrew species in piggeries and found E. bieneusi infections in the three most common rodent species (R. tanezumi, R. norvegicus, and M. musculus). We observed significant differences in prevalence rates among these species, suggesting variations in susceptibility to E. bieneusi (Table 2).

Rattus tanezumi, which is one of the dominant species of house rats in China [16], exhibited the highest prevalence of infection at 23.53% (32/136) in our study. Although this prevalence was significantly greater than the global infection rate of E. bieneusi in rodents (13.6%) [40], it closely matched the previously reported prevalence rate of wild R. tanezumi in Hainan Province, China (23.1%, 31/134) [59], indicating a high susceptibility of R. tanezumi to E. bieneusi. Rattus norvegicus, one of the most common commensal rodent species in the world and dominant in China [16], had an infection prevalence rate of 8.62% (5/58) in our study, which was lower than the prevalence rates observed in wild R. norvegicus in Hainan Province (14.3%, 8/56) [59] and in wild R. norvegicus in six provinces of China (13.3%, 53/399) [29], but similar to the prevalence rates found in wild R. norvegicus from Heilongjiang Province of China (7.9%, 19/242) and wild R. norvegicus from Iran (8.9%, 13/146) [42, 56]. Similarly, Mus musculus, one of the world’s most common commensal rodent species with a wide distribution in China [53], exhibited an infection prevalence rate of 8.00% (2/25) in our study. This rate fell within the range of reported global E. bieneusi infection rates in M. musculus (1.1–28.6%) [6, 29, 31, 36], but was higher than the prevalence observed in wild M. musculus in Slovakia (1.1%, 3/280) and in wild M. musculus from six provinces in China (6.8%, 5/74) [6, 29] and lower than the prevalence observed in wild M. musculus from Germany and the Czech Republic (10.7%, 31/289) and from Poland (28.6%, 6/21) [31, 36]. However, E. bieneusi was not detected in sporadically sampled individuals of A. agrarius (n = 1) or C. shantungensis (n = 7) in our study. Globally, there have been no reports of E. bieneusi infecting C. shantungensis, and the only analysis of E. bieneusi in Apodemus agrarius was carried out in Poland, with a prevalence rate of 42.9% (79/184) [31]. The infection rates of the rodent E. bieneusi show substantial differences in previous studies, potentially affected by factors like host immunity, sample size, experimental methodology, climate, and geographic differences. Therefore, further research is required to identify the factors contributing to this variation.

Table 4 demonstrates that rodents worldwide have been documented with nearly 70 different genotypes of E. bieneusi. In this study, eight genotypes of E. bieneusi were identified, including the zoonotic genotypes D, EbpC, PigEBITS7, and EbpA [20, 23]. The predominant PigEBITS7 genotype was exclusively found in R. tanezumi from two farms, accounting for 23.1% (9/39) of the E. bieneusi isolates. Originally found in pigs in Massachusetts, USA, it was subsequently identified in immunocompromized patients in Jiangxi and Henan in China, as well as in monkeys, wild rats, farmed bamboo rats, and wastewater in China [3, 20, 24, 47, 58]. Notably, this particular genotype has not been found in farmed pigs in China. Therefore, rodents infected with this genotype may be more closely associated with humans and other animal hosts on farms, but still pose a potential risk of infection in farmed pigs. Genotype D, which accounted for 20.5% (8/39) of the E. bieneusi isolates, was found in R. tanezumi and R. norvegicus from four pig farms. This genotype, widely prevalent in rodents, has also been linked to human infections in over 20 countries and has been isolated from more than 25 domestic and wildlife animals, as well as water sources, making it one of the most common E. bieneusi genotypes with a high risk of zoonotic transmission [20, 23, 35, 41, 59].

Genotype EbpC was detected in R. tanezumi, R. norvegicus, and M. musculus from seven pig farms, representing 20.5% (8/39) of the E. bieneusi isolates. As far as we are aware, this is the first documentation of this genotype being identified in M. musculus and R. tanezumi. Notably, this genotype is commonly found in humans, domestic and wild animals, and water sources worldwide [20, 23, 35]. Interestingly, it has been identified as the most prevalent zoonotic genotype in farmed pigs in China [41, 49] and exhibits the broadest distribution in our study area. Additionally, a survey on porcine intestinal parasites conducted on pig farms within our sampling area revealed a 9.5% detection rate of E. bieneusi, with 133 out of 1402 pig fecal samples collected testing positive. Among the positive samples, 54.1% (72 out of 133) had the EbpC genotype (unpublished data). We therefore hypothesized that this finding provides potential evidence of cross-species transmission between farmed pigs and sympatric rodents, but the possibility of other routes of transmission should also be considered, e.g., the possibility that pigs and rodents in the same area may share contaminated food, water, and environment, which needs to be confirmed by further investigation.

Genotype EbpA has been detected in various rodent species worldwide, including R. norvegicus, R. tanezumi, M. musculus, Myocastor coypus, Atherurus macrourus, and Rhizomys sinensis (Table 4) [29, 36, 44, 53, 59]. In our study, it was exclusively identified in R. tanezumi from one pig farm, accounting for 5.1% (2/39) of the E. bieneusi isolates. Moreover, this genotype has been observed in a wide range of hosts, including humans, nonhuman primates, domestic animals (cattle, buffalo, horses, sheep, and goats), pets (dogs), wildlife (deer, foxes, raccoons, bears, pandas, and otters), and birds (pigeons, cranes, and parrots) [20, 23, 28, 37]. It has also been detected in river water and wastewater treatment plants [15, 19, 35, 52]. Thus, the potential cross-species transmission of genotype EbpA poses a zoonotic risk to humans and other animals, while sympatric rodents may act as reservoir hosts for EbpA during the transmission of E. bieneusi.

Four new genotypes of E. bieneusi were identified in this study through sequence analysis of the ITS regions of the obtained E. bieneusi isolates. These novel genotypes were classified into group 1 through phylogenetic analysis and showed one or two base substitutions compared with known zoonotic genotypes, indicating a high risk of zoonotic transmission [20]. Consequently, sympatric rodents infected with E. bieneusi could potentially serve as reservoirs of transmission to humans and other animals, posing a potential threat to public health and ecological security. Additionally, given that farmed pigs are the main hosts of human pathogenic E. bieneusi [20, 23, 41], sympatric rodents inhabiting pig farm environments may facilitate the spillover transmission of this pathogen from pig farms. This is because these rodents are common shuttles between human and animal hosts, as well as between domestic and natural environments. Therefore, controlling rodent populations in the surveyed areas and raising awareness among local populations about the risk of transmission of E. bieneusi from rodents to humans are necessary measures to reduce the threat to public health.

Conclusions

Enterocytozoon bieneusi infection is prevalent in wild sympatric rodents inhabiting pig farm environments in China, with the zoonotic genotype EbpC showing the broadest distribution. The study identified all genotypes as belonging to group 1, indicating that sympatric rodents serve as natural reservoirs for E. bieneusi, posing a potential risk to public health and ecological stability. These findings provide possible evidence for cross-species transmission between sympatric rodents and domestic pigs, implying that sympatric rodents may facilitate the spillover of E. bieneusi from pig farms. Further molecular epidemiological investigations using a One Health approach should be conducted to assess the role of these sympatric rodents in E. bieneusi transmission and explore possible transmission routes in the survey area.

a

These authors contributed equally to this work.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2022YFD1800200), the Henan Province Key Research and Development Plan Project (231111111500), and the National Pig Industry Technology System (CARS-35).

Conflicts of interest

The authors declare that there are no conflicts of interest.

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Cite this article as: Shan F, Meng Q, Wang F, Zhao J, Xu H, Wang N, Liu Y, Zhang S, Zhao G & Zhang L. 2024. Wild sympatric rodents inhabiting pig farm environments may facilitate the spillover of Enterocytozoon bieneusi from pig farms. Parasite 31, 59.

All Tables

Table 1

Prevalence and genotypes of Enterocytozoon bieneusi among rodents and shrews inhabiting pig farms in China.

In the text
Table 2

Risk factors associated with the prevalence of Enterocytozoon bieneusi among rodents and shrews inhabiting pig farms.

In the text
Table 3

Nucleotide variations in the ITS gene region of newly identified Enterocytozoon bieneusi isolates in this study.

In the text
Table 4

Prevalence and genotype distribution of Enterocytozoon bieneusi in rodents in different countries or areas.

In the text

All Figures

thumbnail Figure 1

Map of the sampling areas and the geographical distribution of Enterocytozoon bieneusi detected in the present study. The sampled pig farms are represented by circles on the map and the numbers next to the circles represent farm numbers. The numbers of E. bieneusi genotypes on the surveyed pig farms are indicated by colored circles of different sizes.
In the text
thumbnail Figure 2

Prevalence rates and frequency of Enterocytozoon bieneusi genotypes in rodents and shrews on pig farms. (A) Prevalence rates of E. bieneusi genotypes in this study. (B) Frequency of E. bieneusi genotypes on pig farms.
In the text
thumbnail Figure 3

Phylogenetic relationships of Enterocytozoon bieneusi genotype isolates in this study. Based on sequence analysis of the ITS region, the neighbor-joining method and the Kimura-2 parameter model were used to analyze relationships. Known and novel genotypes are indicated by filled squares and triangles, respectively. Bootstrap values (>50) are indicated at the nodes.
In the text

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