Molecular Detection of Microsporidia in Rabbits (Oryctolagus cuniculus) in Tenerife, Canary Islands, Spain

Simple Summary Microsporidia are a group of fungal-related pathogens widely distributed in the environment, with some species having a negative impact on animals and public health. The European rabbit (Oryctolagus cuniculus) is considered the natural host of Encephalitozoon cuniculi, a microsporidian pathogen of mammals, including humans. The infection caused by E. cuniculi, encephalitozoonosis, ranges from asymptomatic to severe lesions in rabbits, with clinical signs involving the central nervous system, kidney, and eye being the most common. The majority of reported cases have been in domestic rabbits, while cases in wild rabbits are uncommon. Due to the lack of data on microsporidia in the Canary Islands, the aim of this work was to analyze the prevalence and identity of microsporidia in fecal samples from rabbits in Tenerife. Abstract Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidia with zoonotic potential that have been identified in humans, as well as in a large group of wild and domestic animals. Several wildlife species have been studied as reservoirs of zoonotic microsporidia in mainland Spain, including the European rabbit (Oryctolagus cuniculus). Due to a lack of data on microsporidia infection in wildlife on the Canary Islands, the aim of this work was to analyze the prevalence and identify the species of microsporidia in rabbits in Tenerife. Between 2015 and 2017, a total of 50 fecal samples were collected from rabbits in eight municipalities of Tenerife, Canary Islands, Spain. Seven of the fifty samples (14%) were amplified using 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. Sanger sequencing reveals the presence of Encephalitozoon cuniculi genotype I in two samples (4%), and undescribed microsporidia species in five samples (10%). This study constitutes the first molecular detection and genotyping of E. cuniculi in rabbits in Spain.


Introduction
The European rabbit (Oryctolagus cuniculus) is one of the most successful invasive mammals. Ancestors from its native Iberian range have been introduced to every continent except Antarctica and over 800 different islands or island groups, although with mixed success [1]. This lagomorph was introduced to the Canary Islands, a Spanish archipelago located in northwest Africa (13 • 23 -18 • 8 W and 27 • 37 -29 • 24 N), in around the 15th century. This species is distributed throughout the archipelago, and has become a highly sought-after hunting resource [2]. In 2017, rabbit abundance was estimated at a mean value of 2.22 individuals/ha in Tenerife, with a standard deviation of 2.25 individuals/ha, suggesting that there is high spatial variability in the abundance of the species. In general, Biology 2022, 11, 1796 3 of 13 rabbits during the legal hunting season or were collected by laboratory personnel on wild rabbit farms.

Study Area, Sample Collection, and Preparation
The study was conducted in eight municipalities of Tenerife, Canary Islands, between 2015 and 2017 ( Figure 1). A total of 50 fecal samples from wild rabbits were collected. The origin of the samples was: donated by hunters (n = 18); samples from rabbits found dead (n = 5); fresh environmental fecal samples (n = 7); and samples from wild rabbits temporarily housed on an "industrial farm" (n = 11) and "family farms" (n = 9). The industrial farm was located in Granadilla de Abona and the two family farms were located in La Matanza de Acentejo and Tegueste.

Ethical
Considering the work is based on fecal samples, no ethical approval was required for the described study. The fecal samples were donated by hunters that hunted wild rabbits during the legal hunting season or were collected by laboratory personnel on wild rabbit farms.

Study Area, Sample Collection, and Preparation
The study was conducted in eight municipalities of Tenerife, Canary Islands, between 2015 and 2017 ( Figure 1). A total of 50 fecal samples from wild rabbits were collected. The origin of the samples was: donated by hunters (n = 18); samples from rabbits found dead (n = 5); fresh environmental fecal samples (n = 7); and samples from wild rabbits temporarily housed on an "industrial farm" (n = 11) and "family farms" (n = 9). The industrial farm was located in Granadilla de Abona and the two family farms were located in La Matanza de Acentejo and Tegueste.
The samples from farmed rabbits were collected from cages containing 1-3 rabbits per cage. For each sampled rabbit, information including gender, location, and health status was recorded when possible (Supplementary Material, Table S1).
After collection, the samples were placed into sterile plastic containers until delivery to the laboratory, and then deposited in vials containing 2.5% aqueous (w/v) potassium dichromate (K2Cr2O7) solution. The samples were stored at 4 °C until the analysis. The samples from farmed rabbits were collected from cages containing 1-3 rabbits per cage. For each sampled rabbit, information including gender, location, and health status was recorded when possible (Supplementary Material, Table S1). After collection, the samples were placed into sterile plastic containers until delivery to the laboratory, and then deposited in vials containing 2.5% aqueous (w/v) potassium dichromate (K 2 Cr 2 O 7 ) solution. The samples were stored at 4 • C until the analysis.

DNA Extraction
DNA from~500 µL of each fecal sample was extracted using the commercial FastDNA ® Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer's instructions, with the homogenizer FastPrep-24TM 5G (MP Biomedicals, Solon, OH, USA) used as the spore disruptor.

PCR Amplification
A nested PCR was performed in an XP Cycler (Bioer Technology, Hangzhou, China) using generic microsporidia primers described by Katzwinkel-Wladarsch et al. [31], amplifying the partial sequence of the 16S rRNA gene, the whole internal transcribed spacer region (ITS), and the partial sequence of the 5.8S rRNA gene.
The For the second PCR, the mixture was identical except that secondary primers (MSP3, MSP4A, and MSP4B) and 1 µL of primary PCR product were used. Each PCR reaction was then subjected to 35 cycles of denaturation at 94 • C for 45 s, annealing at 54 • C for 45 s, and extension at 72 • C for 1 min, with an initial denaturation at 94 • C for 3 min and a final extension step at 72 • C for 7 min [32].

Sequencing and Sequencing Data Analysis
The nested PCR products with sizes ranging from 300 to 500 bp were sequenced at Macrogen Spain, with the secondary primers in both senses.
The sequences obtained using the Sanger method were interpreted with the MEGA X software [33], subsequently analyzed with the basic local alignment search tool (BLAST), and the identity confirmed by homology comparison.
Studies on microsporidia infection in wild European rabbits are scarce. To our knowledge, microsporidia infection is confirmed in only six studies, four of which are based on serological assays, while the other two studies employ molecular techniques (Table 2).
The estimated prevalence of microsporidia infection depends on the diagnostic method employed, the type of sample, and the host habitat.
Direct methods, such as PCR, are suitable for the detection of microsporidia in an active infection with spore shedding, but could lead to underestimating the prevalence due to intermittent spore shedding periods, such as at the beginning of the primary infection or in chronic infections [9].
Considering the type of sample, the shedding of microsporidia spores in urine appears to be more common than in feces, as reported in the studies carried out by Valencakova et al. [28] and Kimura et al. [76]. The brain and kidney are the most frequently parasitized organ by E. cuniculi [66,67], followed by lung, heart, liver, and intestine. In this work, only fecal samples were analyzed, thus, the prevalence of E. cuniculi could be underestimated.
Enterocytozoon bieneusi and E. intestinalis are often detected in rabbit feces (Table 2), but the absence of these two species may be explained by the low prevalence observed in rabbit populations in Spain and the limited sampling available for analysis in this study (Table 2).
With respect to the host habitat, a higher prevalence has been observed in farmed rabbits, possibly due to the poor hygiene and overcrowding that is often found when rearing rabbits at commercial farms [15,87]. This is in agreement with the results obtained in this work, where E. cuniculi is only detected in wild rabbits kept temporarily on farms, with no positive results found in wild hunted rabbits or in environmental fecal samples. All sampled rabbits were wild-raised, but some of them were temporarily housed on industrial (n = 11) or family farms (n = 9) at the time of sampling, which could have been a risk factor for acquiring the E. cuniculi infection.
The origin of E. cuniculi infection in rabbits in Tenerife is unknown. Despite the limited sample size, the prevalence of E. cuniculi obtained in fecal samples in this study (4%; 2/50) is similar to that found in fecal samples from pet rabbits in China (5.8%; 34/584) [17] and from farmed rabbits in Japan (5.6%; 6/107) [76] using PCR.
A retrospective study carried out between 2000 and 2018 in northern Spain identified encephalitozoonosis, using histology, as the most frequent parasitic disorder found in domestic rabbits, while no cases were observed in wild rabbits [43]. With regard to previous data, the domestic (farmed and pet) rabbit population can be considered a reservoir of E. cuniculi infection in Spain, in contrast to the wild rabbit population.
Undetermined microsporidia species were detected in 5 out of the 50 (10%) rabbit samples from Tenerife. The primers used in this work were not specific and amplified the SSU rDNA of a wide range of microsporidia species. The novel microsporidian sequences detected in the rabbit feces may belong to microsporidia that pass through the digestive system with food or water, as was previously suggested for similar "orphan" sequences discovered in humans [88] and animals [29].
The sequence MicC80 (GenBank accession number OP555069) shares homology with sequences of Bryonosema plumatellae and Schroedera airthreyi, both isolated from a freshwater bryozoan of the genus Plumatella sp. [94,95]. The correlation of less than 85% and the low query cover value with the closest known species suggest that it is an undescribed genus or family of microsporidia [93].
Although the potential for the wild European rabbit to be a zoonotic source of microsporidia infection is relatively low [6,19], the domestic (farmed or pet) rabbit should be considered a source of human pathogenic microsporidia, especially for animal owners and farm keepers. Encephalitozoon cuniculi genotype I has been previously detected in several wild and farmed mammals and birds [24], as well as in immunocompromised [96] and immunocompetent humans [97]. Therefore, the transmission of this infection could pose a risk to public and veterinary health.

Conclusions
This study provides molecular data on microsporidia infection in rabbits in Tenerife, Canary Islands, Spain. The overall prevalence of microsporidia was 14.0%, with five cases of undetermined microsporidia species and two cases of E. cuniculi, all detected in fecal samples.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biology11121796/s1, Table S1: Data of the rabbit samples analyzed in this study.