High Prevalence of Microsporidia in the North African Hedgehog (Atelerix algirus) in the Canary Islands, Spain

Simple Summary It is well known that mammals can harbor various pathogens that can affect humans (known as zoonotic pathogens) including viruses, bacteria, fungi and parasites. Microsporidia are a group of pathogens related to fungi and parasites of several animals that can cause diarrhea or systemic infection in humans. Due to the limited knowledge about microsporidia infection in hedgehogs worldwide, this study aimed to analyze the presence and identity of microsporidia in a group of North African hedgehogs from the Canary Islands (Spain). Enterocytozoon bieneusi and Encephalitozoon cuniculi, two zoonotic species of microsporidia, were identified. These results suggest that microsporidia species with zoonotic risk circulate in the archipelago. Abstract Microsporidia are unicellular eukaryotic obligate intracellular parasites with a wide range of hosts reported worldwide; however, little is known about the epidemiological data on microsporidia infection in animals from the Canary Islands. Since data on microsporidia infection in hedgehog species are scarce, the aim of this study was to analyze the presence and identity of microsporidia in a group of North African hedgehogs (Atelerix algirus) using microscopic and molecular methods. From December 2020 to September 2021, a total of 36 fecal samples were collected from naturally deceased hedgehogs from Tenerife and Gran Canaria. All samples showed spore-compatible structures (100%; 36/36) under microscopic analysis, of which 61.1% (22/36) were amplified via the nested-polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. After Sanger sequencing and ITS analysis, Enterocytozoon bieneusi was detected in 47.2% (17/36) of the samples, identifying two novel genotypes (AAE1 and AAE2), followed by the detection of an undetermined species in 8.3% (3/36) and Encephalitozoon cuniculi genotype I in 5.6% (2/36) of the samples. This study constitutes the first report of microsporidia species in Atelerix algirus worldwide, highlighting the high prevalence of zoonotic species.


Introduction
Microsporidia are unicellular eukaryotic obligate intracellular parasites, spore-forming, and phylogenetically related to the fungi kingdom. Seventeen species have been described All fecal samples were placed in tubes containing 2.5% (w/v) aqueous potassium dichromate solution (K2Cr2O7) (Merck, Darmstadt, Germany) and stored at 4 °C until further processing.  [15] and microscopically screened for microsporidia spores at a magnification of 1000× under a Leica DM750 microscope model ICC50 HD (Leica Microsystems, Heerbrugg, Switzerland). Samples with spore-compatible structures, ovoid and refractile structures stained pink-red, were considered positive.

DNA Extraction
The total DNA of each fecal sample was extracted using~500 µL of the sample, previously washed with sterile Phosphate-Buffered Saline (PBS) 1X at room temperature, and centrifuged at 3500 rpm for 15 min to remove the potassium dichromate solution. A commercial FastDNA ® Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) was used following the manufacturer's instructions, and the homogenizer FastPrep-24 TM 5G (MP Biomedicals, Solon, OH, USA) was used as the spore disruptor.

Nested-PCR Amplification
Nested-PCR was carried out in an XP Cycler (Bioer Technology, Hangzhou, China) targeting the partial sequence of the 16S rRNA gene, the complete internal transcribed spacer region (ITS), and the partial sequence of the 5.8S rRNA gene [16].
Ten microliters of each PCR product were examined via electrophoresis on 1.5% (w/v) agarose gels (Fisher Bioreagents, Madrid, Spain) stained with REALSAFE Nucleic Acid Staining Solution (20,000×, REAL, Durviz S.L., Valencia, Spain). An amplified DNA product with sizes between 300 and 500 bp (expected size for Encephalitozoon spp. and E. bieneusi, respectively) was considered positive and was sequenced using secondary primers.

Sequencing and Phylogenetic Analysis
All nested-PCR positive products were purified using ExoCleanUp FAST (VWR International, Haasrode, Belgium) and sequenced using the Sanger method at the University of La Laguna Sequencing Services (Servicio de Genómica-Servicios Generales de Apoyo a la Investigación de la Universidad de La Laguna, Universidad de La Laguna, Spain).
The obtained sequence chromatograms were analyzed and aligned using the ClustalW program included in MEGA X v10.2.6 (Molecular Evolutionary Genetic Analysis) software (Hachioji, Japan) [18] and compared using the Basic Local Alignment Search Tool (BLAST) in the GenBank database.
Phylogenetic trees were generated using the neighbor-joining method, and genetic distances were calculated using the Kimura 2-parameter model [19,20] with 1000 bootstrap replicates.

Light Microscopy
Of the 36 hedgehogs, spore-compatible structures were found in 100% (36/36) of the samples stained with Weber's chromotrope stain. undetermined species based on BLAST analysis because of the low homology observed (less than 95%) or because they were not long enough for homology comparison with the reference sequences in MEGA X.
The ITS region of genotype AAE1 was 242 bp in length, as a result of the deletion of one nucleotide (position 53), differed by one single nucleotide polymorphism (SNP) compared with genotype HND-I, two SNPs compared with genotype EA1, one SNP and two nucleotide insertions compared with genotype EA2, two SNPs compared with genotype EA3, one SNP compared with EA4, and two SNPs compared with genotype S9. The positions of the SNPs and the insertions are listed in Table 1.
The ITS region of genotype AAE2, 243 bp in length, showed only one SNP at position 104 (G→ A) compared to genotype WildBoar3.
The novel genotypes clustered within Group 1 based on phylogenetic analysis ( Figure 2). Genotype AAE1 fell into an independent clade close to two clades, one of which was formed by the genotypes isolated from the Amur hedgehog (E. amurensis) in China (EA1-EA4) (bootstrap value of 100%). Genotype AAE2 clustered in a clade with the genotype Wild-Boar3 (syn. NCF2, NCF3, NCF4), which have been previously detected in carnivores in mainland Spain.

Molecular Characterization of Encephalitozoon cuniculi
Two of the thirty-six (5.6%) fecal samples were positive for E. cuniculi. Both sequences showed >99% homology with E. cuniculi sequences deposited in GenBank (AB713183.1, L13332.1, and OP555067.1). Phylogenetic analysis confirmed the identity of the isolates as E. cuniculi (bootstrap 100%) ( Figure 3) and were identified as genotype I based on ITS sequence analysis. which was formed by the genotypes isolated from the Amur hedgehog (E. amurensis) in China (EA1-EA4) (bootstrap value of 100%). Genotype AAE2 clustered in a clade with the genotype WildBoar3 (syn. NCF2, NCF3, NCF4), which have been previously detected in carnivores in mainland Spain.

Figure 2.
Phylogenetic relationships between the sequence of the internal transcribed spacer region of the rRNA gene of Enterocytozoon bieneusi obtained in this study and the sequences of known genotypes deposited in GenBank. The tree was constructed using the neighbor-joining method based on the genetic distance calculated using the Kimura 2-parameter model. Representative sequences from each phylogenetic Group of E. bieneusi genotypes (Groups 1-11) were used. Accession numbers are shown in bold, and information regarding the host species and origin are shown in parentheses. There were a total of 239 positions in the final dataset.

Molecular Characterization of Encephalitozoon cuniculi
Two of the thirty-six (5.6%) fecal samples were positive for E. cuniculi. Both sequences showed >99% homology with E. cuniculi sequences deposited in GenBank (AB713183.1, L13332.1, and OP555067.1). Phylogenetic analysis confirmed the identity of the isolates as E. cuniculi (bootstrap 100%) ( Figure 3) and were identified as genotype I based on ITS sequence analysis.

Geographical Distribution
Of the 16 genotyped E. bieneusi isolates, the most frequently detected was genotype

Geographical Distribution
Of the 16 genotyped E. bieneusi isolates, the most frequently detected was genotype AAE1 (81.25%; 13/16). It showed a wide distribution, being detected in seven municipalities in Tenerife and in the sampled municipality in Gran Canaria. In contrast, the genotype AAE2 (18.75%; 3/16) was detected only in two northern municipalities of Tenerife Island, El Sauzal, and Tacoronte.
Encephalitozoon cuniculi-positive hedgehogs were found in the northeastern zone of Tenerife, specifically in Santa Cruz de Tenerife and El Rosario.
Undetermined species were detected in Adeje and San Cristóbal de La Laguna; no other species were detected at these locations ( Figure 1, Table 2). Table 2. Geographical distribution of microsporidia species and genotypes identified in Atelerix algirus on the Canary Islands.

Location
Sample Size (n)

Discussion
The present study constitutes the first report of microsporidia in the North African hedgehog (A. algirus), highlighting the presence of human-pathogenic microsporidia, E. bieneusi and E. cuniculi.
Data on microsporidia infection in patients from the Canary Islands are scarce, with only two reports of E. bieneusi in immunocompetent patients from Tenerife [35] and transplant recipients from Gran Canaria (genotype D) [36].
The prevalence obtained using the microscopic method in this study (100%; 36/36) differed from that obtained using nested-PCR (61.1%; 22/36), as reported in other studies [46,50]. This difference can be explained by the spontaneous extrusion of spores or low parasitic load, as suggested in the studies conducted by Izquierdo et al. [50] and Haro et al. [53], respectively.
The overall prevalence obtained in animals using the molecular methods in studies conducted in Spain with similar sample sizes ranged from 43.8% (14/32) in domestic dogs in Madrid [45], to 55.6% (15/27) in farmed pigs in Extremadura and Castile and León, [45] and 65.4% (17/26) in Iberian lynx in Andalusia [50], with E. bieneusi being most commonly detected in fecal samples in the latter studies. However, E. bieneusi was detected in 7 of the 14 PCR-positive dog samples and in 7 of the 15 PCR-positive pig samples compared to the 17 of the 22 nested-PCR-positive samples detected in this study.
To the best of our knowledge, this is the first study to detect E. cuniculi in hedgehogs. The prevalence obtained (5.6%; 2/36) was similar to that recently reported in fecal samples from European rabbits in Tenerife (4.0%; 2/50) [61]. Genotype I of E. cuniculi has been the only genotype detected in animal hosts in Spain to date [60,61], and the species is less frequent in this country, with a few cases in humans [40][41][42]. However, molecular detection in water sources, in addition to serological analysis of domestic and wild animals, demonstrates the presence of this parasite in the environment [50,61,62].
Considering that the diet of hedgehogs is mainly based on Coleoptera [63] and numerous species of microsporidia have been described as insect parasites [64], the undetermined species detected in fecal samples of hedgehogs are suspected to be microsporidia species infecting invertebrate hosts. Other studies have also detected undetermined species using molecular methods [45,61,62].
The remaining human-pathogenic Encephalitozoon species, E. intestinalis and E. hellem were not detected in this study. A low prevalence of E. intestinalis has been reported in animals in Spain [45,47,53,58] and E. hellem in mammals worldwide [65].
Considering the high prevalence of E. bieneusi genotypes with zoonotic potential, veterinary control measures should be implemented to detect this pathogen, given that hedgehogs have been kept as pets on the Canary Islands [66] and could pose a risk to children who are most susceptible to microsporidiosis [67]. Despite the limited number of E. cuniculi cases detected in this study, the zoonotic risk should not be underestimated because symptomatic cases have been documented in humans [9].

Conclusions
This study constitutes the first report of microsporidia in fecal samples of the North African hedgehog (A. algirus). The overall prevalence of nested-PCR-confirmed samples was 61.1% (22/36), with E. bieneusi being the most common species, followed by the undetermined species and E. cuniculi. Two novel genotypes of E. bieneusi were identified, named AAE1 and AAE2, both clustered within Group 1, and the E. cuniculi isolates were identified as genotype I.
The results obtained in this study provide new data on the epidemiology of microsporidia on the Canary Islands (Spain), suggesting that zoonotic genotypes of humanpathogenic microsporidia circulate in the fauna of the islands, posing a risk to public and veterinary health.

Institutional Review Board Statement:
We consider that the Ethical Declaration is not necessary since in this study only dead or naturally deceased hedgehogs were used.

Informed Consent Statement:
The head of the Wildlife Recovery Center has signed the consent form.
Data Availability Statement: Not applicable.