Introduction

Encephalitozoon spp. is a microsporidian obligate intracellular spore-forming parasite and is considered to be one of the most common microsporidian parasites in humans [1]. Encephalitozoonosis is rare in healthy people but can be a complication in patients with weakened immune systems [2,3,4,5]. The species Encephalitozoon cuniculi, Encephalitozoon intestinalis and Encephalitozoon hellem were detected in various birds and mammals [6] with a high prevalence of Encephalitozoon spp. in some of the domestic animals. Wild animals can also be infected and can serve as reservoirs of several infections including zoonotic diseases [7]. The usage of molecular methods helps to detect and differentiate strains of Encephalitozoon spp.

Didier et al. [8] described 3 different genotypes (I, II, and III) of E. cuniculi strains with different number of 5′-GTTT-3′ repeats in the internal transcribed spacer (ITS) region of the ribosomal RNA gene. More recently, a novel genotype IV was identified in a human patient [9]. The types of E. cuniculi are sometimes referred as the “rabbit strain” for type I, the “mouse strain” for type II, the “dog strain” for type III, and the “human strain” for type IV in association to the host from which they were isolated. Although each strain has a preferential host species, it is known that E. cuniculi has a low host specificity. For example, genotype III (“dog strain”) was detected also in wild small rodents [10, 11]. The main host of E. hellem are humans, but it has been found also in birds. Correspondingly, Mathis et al. [12] identified strain variation of E. hellem based on the sequence of the ITS of the ribosomal DNA (rDNA) showing the ITS sequence seems a valuable marker.

Free-living small mammals, mainly rodents, are known carriers and reservoirs of more than 60 diseases with zoonotic potential. Thanks to their adaptive way of life, they can be found both in the wild and in area heavily populated with humans and thus can represent the risk of infection for domestic animals and human. Since the sylvatic life cycle of Encephalitozoon spp. is not yet fully understood, the aim of the study was to conduct molecular detection and genotype characterization of Encephalitozoon spp. in wild small mammals trapped in localities both near to and at a large distance from residential areas.

Materials and Methods

Sampling

Wild small mammals were trapped using the snap and life traps in 41 different localities of the Czech Republic (Fig. 1) during two trapping sessions (June and September) in the years 2015 and 2016.

Fig. 1
figure 1

Map showing 41 localities of 6 areas (4 areas: Sokolov, Příbram, Roudný, and Helfenburk in Bohemia and 2 areas: Poodří Protected Landscape Area and the Moravian Karst in Moravia) in the Czech Republic, where 300 wild small mammals were trapped for detection Encephalitozoon spp. by nested PCR

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In Bohemia (western part of the Czech Republic), the trapping was done in 36 localities of 4 areas (Helfenburk, Příbram, Roudný, and Sokolov). In Helfenburk and Příbram, the trapping plots were situated near to residential zones, recreational areas, and sports facilities. On the contrary, in Sokolov and Roudný, the trapping plots were located significantly further from residential areas in grasslands, meadows, forests, wetlands, and spoil heaps. At each trapping site, 25 traps were set for 3 consecutive nights in a standard trapping grid or lines (based on the plots’ characteristics) with a 5-metre distance trap-trap.

In Moravia (eastern part of the Czech Republic), the trapping was done in 5 localities of 2 protected landscape areas (the Moravian Karst and Poodří Protected Landscape Area), further from residential areas, in wet meadows, hornbeam, and oak forests. The traps were placed on the ground in a line, with 7-metre spacing between them.

The researchers involved in the sampling were experienced in safety protocols during animal trapping and handling and followed the conditions of the long-term experimental project (Ethical Approval Statement). The basic principles of biosafety to protect persons and the environment were respected. The animals were necropsied in an isolated zone of the laboratory by personnel dressed in laboratory coats, gloves, eye and face protection and 3 M respirators.

In total, 300 trapped animals (274 Rodentia and 26 Eulipotyphla) of different species, sex, and age (adult, sub-adult, and juvenile) were analyzed for the detection of Encephalitozoon spp. (Table 1). Tissue samples (brain or liver) collected during autopsy were frozen at – 20 ºC until DNA isolation.

Table 1 Detection of Encephalitozoon spp. by nested PCR in wild small mammals trapped in the Czech Republic
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Molecular Methods

The DNA was isolated by DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany) following producer instructions. Before that, pieces (≤ 25 mg) of brain or liver (when brain was not available) tissues were homogenized by MagNA Lyser Instrument (Roche, Basel, Switzerland), using ceramic marbles. The samples were examined by nested PCR amplifying rDNA internal transcriber spacer region (ITS), with the use of two pairs of microsporidia-specific MSP1, MSP2A, MSP3 and MSP4A primers [13]. The PCR mixture contained 12 µl of commercial premix PPP master mix (Top-Bio s.r.o., Prague, Czech Republic), 6 µl of PCR grade water, 1 µl of each primer and 5 µl of DNA. A positive control (DNA isolated from spores of E. cuniculi) and a negative control (PCR grade water, Top-Bio s.r.o.) were included in the PCR reaction. The PCR protocol for both steps included an initial denaturation at 92 °C for 2 min, followed by 35 cycles of denaturation at 92 °C for 1 min, annealing at 59 °C for 1 min, and extension at 72 °C for 1.5 min, with a final extension step at 72 °C for 5 min. After electrophoresis on 1.5% agarose gel with Midori Green stain, animals were evaluated to be positive, when tissue (brain or liver) was positive. Positive PCR products were purified (Gel/PCR Fragments Extraction Kit, Geneaid, New Taipei City, Taiwan) and sent for sequencing (Macrogen, Amsterdam, the Netherlands). Final sequences were edited by Staden Package Programs (Pregap4, Gap4) and compared with sequences in GenBank using BLAST (Basic Local Alignment Search Tool).

Statistical Analysis

The results were statistically analysed with Pearson’s chi-square test for independence, using STATISTICA Cz 12 [14]. We tested the null hypothesis that a prevalence of Encephalitozoon spp. does not differ among species, sex, age, and areas of trapping (proximity to residential area). The differences were considered statistically significant if the p-value was < 0.05. In the case of a statistically significant difference of positivity in some of the variables, the Scheffé’s multiple comparison method [14] was subsequently applied to identify a statistically significant difference between pairs of animal species. Subsequently, the odds ratio (OR) of chances for these pairs was calculated. The results were presented as percentages with 95% confidence intervals (CI).

Results

The DNA of Encephalitozoon spp. was proved by nested PCR in 11% (32/300) of animals, with 13% (26/200) in animals trapped in Bohemia and 6% (6/100) in animals trapped in Moravia, without statistical difference (p > 0.05) in these two parts of the Czech Republic (Table 1). The Scheffé’s multiple comparison method showed a statistically significant difference (p < 0.0015) in prevalence of Encephalitozoon spp. between species, specifically in Apodemus flavicollis (1%) and Micromys minutus (50%) (OR = 69. 95% CI: 6.0 -747.7). The prevalence of Encephalitozoon spp. statistically differed (p < 0.05) depending on the areas of trapping, with a higher prevalence (29%, 12/42) in areas close to residential areas, compared to the prevalence (9%, 20/258) in areas at a large distance from residential areas. There was no statistically significant difference (p > 0.05) in prevalence between the sex and age categories of animals.

Analysis of the genotypes by BLAST search in the GenBank database showed the highest homology with E. cuniculi genotype II in 22 of 32 positive samples (1 isolated from Apodemus agrarius, 4 from Apodemus sylvaticus, 8 from Myodes glareolus, 5 from Microtus arvalis, 2 from Micromys minutus, and 2 from Sorex araneus). Sixteen of these samples were from animals trapped in Bohemia (2 from Helfenburk, 4 from Příbram, 2 from Roudný and 8 from Sokolov), and six were from animals trapped in Moravia (3 from The Moravian Karst and 3 from Poodří). One sample isolated from juvenile female A. sylvaticus trapped in Bohemia (Helfenburk) was characterised as Encephalitozon hellem genotype 1 A. Genotyping of 9 samples was not completed.

Discussion

Wild animals living in urban or suburban areas are often monitored as reservoirs of various pathogens. In our study, molecular methods confirmed the presence of Encephalitozoon spp. DNA in the brain or liver of 11% of wild small mammals. In a previous study, the DNA of E. cuniculi was detected by PCR in brain of 6.5% wild small mammals from Austria [15] and affection of the central nervous system was also described in rabbits as one of the most common symptoms of E. cuniculi disease [5, 16]. In other studies from Central Europe, different tissue samples were used for detection of Encephalitozoon spp. For example, Perec-Matysiak et al. [11] examined by nested PCR spleen and faecal samples of wild small mammals trapped in nature reserves and suburban recreational areas in Slovakia, Poland, and the Czech Republic. They found Encephalitozoon spp. in 15% of animals, which was only slightly higher than the positivity in our study. Sak et al. [17] proved Encephalitozoon spp. by nested PCR in faecal samples of 25% wild rodents trapped across the Czech Republic-German border. They showed only a slight correlation between positivity and the proximity to residential areas that is in contrast to our results. Danišová et al. [18] proved E. cuniculi and E. intestinalis by real-time PCR in faecal samples of 0.4% wild small mammals from Slovakia.

The trapping localities in our study had different characteristics in terms of proximity to residential areas, with statistically higher Encephalitozoon spp. positivity in animals trapped in near to residential areas. The most often infected animal species in our study was M. minutus, with all animals of this species having been trapped close to residential areas (proximity to a football stadium and adjacent family houses in Příbram). In Bohemia, Encephalitozoon spp. was detected in 13% of animals trapped in urban areas, whereas a lower positivity of 6% was observed in animals trapped in protected landscape areas in Moravia, where there is less frequent contact with humans or domestic animals.

Based on molecular techniques, the presence of 4 different genotypes of E. cuniculi (genotypes I-IV) was previously confirmed [8, 9]. Genotyping analysis of positive samples from our study showed that 69% (22/32) were E. cuniculi genotype II and 3% (1/32) E. hellem genotype 1 A. E. cuniculi genotype II was also proved in 93% of wild rodents from Slovakia, Poland, and the Czech Republic, while genotypes I and III were proved only in 1.5% and 6% of animals, respectively [11]. In contrast, Sak et al. [17] proved genotype I to be more frequent (58%) compared to genotype II (42%) in wild small mammals from the Czech Republic. Based to the previous studies, it seems that genotype II is more frequent in wildlife reservoirs compared to other genotypes, nevertheless, infection with this genotype was also described in humans e.g. in 8% of renal transplant recipients and in 1% of patients with various respiratory diseases [19]. Despite the fact that E. hellem genotype 1 A is typical for birds [6], this genotype was proved in material from human immunodeficiency virus-positive patients [20] and also in 9% of 2 subspecies of the house mouse trapped in the Czech Republic and Germany near or within residential areas or stables [17]. This points to the fact that host specificity is not strictly limited to birds. Central Europe, and especially the Czech Republic, is among the best explored areas concerning the number of studies focused on the spread of E. cuniculi infection in wildlife [6]. Further research in this country is essential to explain the method of transmission of infection in wildlife, which has not yet been fully understood.

Based on our results, wild small mammals can play a crucial role as a reservoir in the transmission of Encephalitozoon spp. infection. They can spread Encephalitozoon spp. through sympatric rodents to the domestic animals, but they may also spillover the infection from domestic animals [21]. This could pose a threat to domestic animals and to humans, who might become infected with Encephalitozoon spp. from food and water contaminated with spores or through the consumption of infected farm animals. Moreover, according to a laboratory experiment, spores of E. cuniculi genotype II may remain infective even after fermentation at 24 °C for 48 h [22]. This contributes to the risk of alimentary infection in humans after ingestion of uncooked meat products from domestic animals containing spores of E. cuniculi.

It seems that there was a sufficient scope in terms of the diversity of localities with four areas in Bohemia, and two in Moravia including also the area in warmer southern Moravia. A limitation of the study could be the sample size (n = 300) and the result might be partly influenced by the sequencing results, when just 22 of 32 positive samples (69%) were successfully sequenced. The rest positive samples could distort the ratio of occurrence of genotype II to genotype I, however, it is evident that genotype II would prevail.

Conclusion

The Encephalitozoon spp. is parasite present worldwide and circulates in both animal and human populations. Given its high environmental resistance, low host specificity, and potential to cause serious disease or even death in both animals and humans, it is important to study this pathogen within a One Health framework. We proved higher positivity in localities near to residential areas compared with other with a large distance. Even more, genotyping analysis revealed E. cuniculi genotype II and Encephalitozon hellem genotype 1 A. The results of our study brought new information for better understanding the sylvatic life cycle of Encephalitozoon spp.