Molecular characterization and new genotypes of Enterocytozoon bieneusi in minks (Neovison vison) in China

Microsporidiosis is an emerging and opportunistic disease, and Enterocytozoon bieneusi is the main cause of this disease in humans. Little information is available on prevalence and genotyping of E. bieneusi in minks. We collected 559 feces samples of minks from Heilongjiang and Jilin provinces in 2017, and studied E. bieneusi prevalence by nested PCR. A total of 23 out of 559 minks (4.1%) were detected as E. bieneusi-positive, and were raised in five of the seven investigated farms. Age was the only risk factor associated with E. bieneusi prevalence in investigated minks through logistic regression analysis. Sequence analysis of the ITS gene revealed that five E. bieneusi ITS genotypes, including Peru11, EbpC, and three novel genotypes (HLJM-1, HLJM-2 and JLM-1) were present, suggesting minks may be a potential source of human microsporidiosis.


Introduction
Minks are a species of high economic importance: the animals are widely raised for their fur in Northern China, including in Heilongjiang and Jilin provinces. Minks can serve as reservoirs for many pathogens including influenza viruses [7], Aleutian mink disease virus [17], thrombocytopenia syndrome virus [21], Pentatrichomonas hominis [14], and Toxoplasma gondii [30]. Because minks are in close contact with their feeders, they can transmit many pathogens to humans, including Toxoplasma gondii [30]. Despite this, data regarding the prevalence and genotypes of Enterocytozoon bieneusi in minks are scarce.
The Microsporidia contains over 1300 named species, and has a worldwide distribution. Enterocytozoon bieneusi is the most frequent causative agent of human microsporidiosis [1,11] and is responsible for more than 90% of human infections [3]. E. bieneusi can infect a variety of invertebrates and vertebrates [27,28,33], and can be transmitted through the anthroponotic, zoonotic, water-borne, and/or food-borne routes [4,13,19]. The symptoms of microsporidiosis caused by E. bieneusi are diarrhea and abdominal pain in immunodeficient individuals, while the infection appears asymptomatic in immunocompetent individuals who can shed spores into the environment and become a potential source of infection for other individuals [29].
More than 240 E. bieneusi genotypes have been defined based on the internal transcribed spacer (ITS) region of the rRNA gene [1]. All the genotypes can be grouped into 9 groups (named groups 1-9). The majority of human infections are caused by the zoonotic group 1 [6,15]. However, some genotypes (such as I, J and BEB4) from the other zoonotic groups have also been found in humans [8].
In order to determine whether minks can be infected by E. bieneusi, and to assess the zoonotic risk of E. bieneusi between minks and humans, a total of 559 mink feces samples were collected from seven farms in Heilongjiang and Jilin provinces. The samples were tested to detect the prevalence of E. bieneusi and associated genotypes in minks by nested PCR amplification of the ITS region of E. bieneusi.

Ethics statement
All animals were handled in strict accordance with good animal practices according to the Animal Ethics Procedures and Guidelines of the People's Republic of China, and the study was approved by the Ethics Committee of Jilin Agricultural University.

Specimen collection
In all, 559 farmed mink fecal samples were randomly collected from Heilongjiang (43°26 0~5 3°33 0 N, 121°11 01 35°05 0 E) and Jilin (41°~46°N, 122°~131°E) provinces, northeastern China in 2017. More than 200 minks were bred at each farm, and the sampling percentage ranged from 5% to 10% on the different farms. Fresh dejections were immediately collected using a polyethylene glove, and were then stored in ice boxes and transported to the laboratory. The Farm ID, gender and age of minks were obtained from the owners.

DNA extraction and PCR amplification
The commercial E.Z.N.A. Ò Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) was used to extract genomic DNA, following the manufacturer's instructions, and extracted DNA was stored at À20°C. PCR targeting the ITS region was used to explore the prevalence and genotypes of E. bieneusi. All the PCR operations have been described in a previous study [31]. In each trial, positive and negative controls were present. The amplification products were observed using UV light after electrophoresis in a 1.5% agarose gel containing GoldView TM (Solarbio, Beijing, China).

Sequence and phylogenetic analyses
Sangon Biotech Company (Shanghai, China) was contracted to sequence the PCR products. Sequence accuracy was evaluated by bidirectional sequencing. Replicates were made when new sequences were found (single nucleotide substitutions, insertions or deletions). ClustalX 1.83 was used to align the sequences. The neighbor-joining (NJ) method (Kimura 2-parameter model, 1000 replicates) was used to reconstruct the phylogenetic trees with Mega 5.0 software. Representative nucleotide sequences were deposited in Gen-Bank under accession numbers MH052578-MH052582.

Statistical analysis
Data analysis of the prevalence of E. bieneusi infection in minks by age, gender, and different farms groups was performed by v 2 testing using SAS version 9.1 (SAS Institute, Cary, NC, USA) [16,32]. When p < 0.05, the results were considered statistically significant. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were estimated to explore the strength of the association between E. bieneusi-positivity and the conditions investigated.
The ITS sequence analysis demonstrated that the sequence of Accession Nos. MH052578 and MH052582 was identical to that of genotypes Peru11 (Accession No. KT922238) and EbpC (Accession No. KX905207), respectively. Moreover, all the five identified E. bieneusi genotypes were grouped into group 1 (Fig. 1). Peru11 was sub-classified into group 1a (Fig. 1); EbpC, HLJM-1, HLJM-2 and JLM-1 were sub-classified into group 1d (Fig. 1). More importantly, Peru11 and EbpC were also found in HIV-positive patients in Henan [20]. These findings suggest that minks may play an important role in human infections. Although no evidence of human microsporidiosis outbreaks originating from minks or other animals has been found, we should pay close attention to nosocomial transmission among humans, minks and other animals.