First report on molecular identication of Eimeria sp. from captive forest musk deer

Background: Forest musk deer (Moschus berezovskii) is a national rst-level protected and endangered wild animals in China. The intestinal coccidiosis of captive forest musk deer is one of the most important diseases. However, few studies have been conducted to quantify Eimeria sp. infection and to identify its molecules. Thus, the objective of this study was to investigate the Eimeria sp. infection in the intestinal tract of forest musk deer in Sichuan and Shaanxi, China, and to identify the 18S rRNA gene fragment of Eimeria sp. , which provides scientic basic experimental data for the molecular epidemiological investigation and population genetic analysis of Eimeria sp. of captive forest musk deer in 7 regions. Methods: 328 faecal samples of forest musk deer were collected from 7 farms. The DNA of Eimeria sp. in the positive samples was extracted and used as template for nested PCR amplication. The 18S rRNA gene fragment was connected with the plasmid vector, and the products were introduced into Escherichia coli (DH5α). The culture bacterial solution was used as a PCR reaction template for identication. Results: In total, we collected 328 faecal samples from forest musk deer in Lixian (n=54), Maoxian (n=52), Ma’erkang (n=49), Dujiangyan (n=55), Hanyuan (n=41), Luding (n=36) and Weinan (n=41). 198 (60.37%) faecal samples were positive for Eimeria sp. We analyzed the 18S rRNA gene sequence of Eimeria sp, and determined 34 types with similarity of 90.5% ~ 100%. A phylogenetic tree constructed based on the 18S rRNA gene sequence of Eimeria sp., it was conrmed that Eimeria sp. parasitized in the intestinal tract of forest musk deer was closely related to Eimeria alabamensis from Bos taurus, Eimeria faurei from Ovis aries and Eimeria ahsata from Ovis aries. Conclusions: To our knowledge, this was the rst molecular identication


Background
Forest musk deer (Moschus berezovskii) is a medium-sized mammal that inhabits alpine forests [1,2]. The forest musk (Moschus spp.) is an endangered species in China and is currently listed as a class I protected species at the national level [3,4,5]. Musk is secreted by the musk gland in the groin of adult male forest musk deer, it has high economic and medicinal value [6,7]. In recent years, due to habitat destruction and other reasons, the population of wild forest musk deer has decreased sharply [4].
Therefore, China began to carry out research on arti cial breeding forest musk deer in Sichuan, Shaanxi and other regions [8,9]. However, the requirements of forest musk deer on breeding conditions are strict [10,11], and the prevention and control technology for herd diseases of captive musk deer is weak. The actual number of captive forest musk deer is still very small, and large-scale breeding of ruminants similar to cattle and sheep has not been successfully realized [12].
Coccidia have been described in a diverse range of vertebrate host groups [13,14]. Several species cause signi cant mortality or morbidity in some hosts, which has attracted special attention in animal production industries [15,16,17]. In ruminants the genus Eimeria sp. is commonly referred to by the term coccidia. Eimeria sp. species are generally gastrointestinal parasites, and most species of Eimeria sp. are exclusively located in the intestine [18]. Coccidiosis is widespread among ruminants [19], the intestinal coccidiosis of captive forest musk deer is one of the important mass diseases, especially endangering young animals, which causes the decrease of feed utilization rate, growth rate, production performance, and musk production capacity [20]. The forest musk deer that are parasitized by Eimeria sp. are emaciated, anaemic and have intestinal in ammation [18,21,22]. Sha et al. [23] found two kinds of forest musk deer coccidia, and named them Eimeriamoschus and Eimeriajinfengshanenisis. Afterwards, they were detected in captive forest musk deer farms in Sichuan and Shaanxi, with a high positive rate of infection [24,25]. Zhao et al. [25] detected 7 species of Eimeria sp. from 50 fresh feces of captive forest musk deer collected from the musk deer farm of Chongqing Institute of Drug Cultivation. The species of coccidia eggs were identi ed as Eimeria stiedai, Eimeria perforans, Eimeria magna, Eimeria media, Eimeria irresidua, Eimeria piriformis and Eimeria coecicola. Lu et al. [26] conducted an investigation on intestinal parasite infection of wild musk deer feces collected in Qinghai Province, of which the infection rate of Eimeria sp. coccidia was 43.66% (31/71). So far, there was no report on the molecular identi cation of Eimeria sp. parasitized on forest musk deer, this study aimed to provide scienti c basic experimental data for the molecular epidemiological investigation and population genetic analysis of Eimeria sp. of captive forest musk deer in 7 regions.

Fecal sample collection
In the spring and autumn of 2018, 328 faecal samples of forest musk deer with clinical symptoms of emaciation, diarrhea, lumpy or unformed stool, listlessness and yellowish fur were collected from 7 forest musk deer farms in Lixian, Maoxian, Ma'erkang, Dujiangyan, Hanyuan, Luding and Weinan of China. The speci c sampling information about faecal samples of captive forest musk deer was as follows (Table 1). At 7: 30 in the morning, fresh faecal samples were collected using sterile disposable PE gloves, put into sterile individual plastic bags and marked with numbers, and recorded the sampling place, time, longitude, latitude and altitude. Finally, the collected samples were placed on ice bag in containers, and then directly brought back to the Animal Quarantine Laboratory of Sichuan Agricultural University for inspection. Parasitological Examination All faecal samples of captive forest musk deer were tested for parasite eggs or oocysts by saturated saline oating method, and the eggs or oocysts found were judged to be positive. The mean eggs per gram (EPG) or oocysts per gram (OPG) of parasites in fecal samples were counted using the McMaster technique [27]. The eggs and oocysts were examined and photographed under a microscope at 400×, and recorded the number of observed coccidian eggs or oocysts [21].

Dna Extraction
Faecal samples were washed with double steaming water and vortexing for 2 min at 12000 rpm. This process was repeated three times until the supernatant was clear. Genomic DNA was then extracted from approximately 200 mg of each semi-puri ed product, using the stool DNA Kit (TD601; Tianmo, Beijing, China). DNA samples were stored in 200 µL of the kit Solution Buffer at -20 °C until use.

Nested Pcr Ampli cation And Amplicon Sequencing
The extracted DNA sample was used as template, a region of the 18S rRNA gene (1500 bp) was ampli ed with the forward primer EF1 5'-GAAACTGCGAATGGCTCATT-3' and the reverse primer ER1 5'-CTTGCGCCTACTAGGCATTC-3' [28]. PCR was performed in a 20 µL volume containing 10 µL 2 × Taq PCR Master Mix (Qingke, Beijing, China), 6 µL deionized water (Qingke), 2 µL DNA, and 1 µL each of forward and reverse primers. Reaction cycles consisted of an initial denaturation step at 95℃ for 5 min, 36 cycles of denaturation at 95℃ for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min 30 s, and a nal extension at 72 °C for 10 min. The second PCR was conducted with the rst PCR ampli cation mixture as the template. The target gene (830 bp) was ampli ed with the forward primer EF2 5'-TTTGATGGTCATTTTTAC-3' and the reverse primer ER2 5'-AATCCTTCTTATGTCTGG-3'. Second reaction system was the same as the rst, thermocycling for target gene was done with an initial denaturation step at 95℃ for 5 min, 32 cycles of denaturation at 95℃ for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 45 s, and a nal extension at 72 °C for 10 min. PCR products were subjected to 1% agarose gel electrophoresis [11]. The 18S rRNA target gene fragment was puri ed and placed in a T100 thermal cycler Bio-Rad together with plasmid pMD19-T vector (Katara, Beijing, China) for overnight connection at 16℃. The products were introduced into Escherichia coli (DH5α) (Tiangen, Beijing, China). Positive colonies were selected and inoculated with the Amp-containing liquid LB medium. The culture bacterial solution was used as a PCR reaction template for identi cation (Youkang, Hangzhou, China).

Data Analysis
The content of each base (A, T, G, and C) in the sequence was calculated by MEGA 6.0 software. The 18S rRNA sequences of different types of Eimeria sp. were compared with 18S rRNA sequence information of other protozoa retrieved ( Table 2) from GenBank in NCBI (https://www.ncbi.nlm.nih.gov/guide/), and DNAMAN 6.0 software was used for sequence alignment analysis, moreover mutation sites in gene sequences were counted. Sequence similarity analysis was performed using DNAMAN 6.0 and Megalign in DNASTAR.Lasergene.v7.1. The 18S rRNA gene sequence of Isospora ohioensis was taken as the outer group of the phylogenetic tree. The software MEGA6.0 neighbor-joining method was used to construct the phylogenetic tree, and tree reliability was determined using bootstrap analyses of 1000 replicate.  (Fig. 2).

Pcr Identi cation Of 18s Rrna Gene Clone
The template of Eimeria sp. 18S rRNA gene clone was identi ed by routine PCR. The results of PCR products detected by agarose electrophoresis are as follows (Fig. 3).

Molecular Identi cation Results
After  Table 3. The 18S rRNA gene sequences of 34 types of Eimeria were compared and analyzed in this experiment. The results showed that the intraspeci c similarity was very high (Fig. 4) The 18S rRNA gene sequence of Eimeria sp. determined in this study was located in two branch systems respectively, and it was under the same branch with Eimeria alabamensis from Bos taurus and Eimeria faurei from Ovis aries, which indicated that they were closely related. DJY3 and Eimeria ahsata were in the same branch (Fig. 5) Table 3)

Discussion
Compared with other countries, China's forest musk deer breeding is obviously more successful [29]. However, the incidence of intestinal diseases and mortality rate of forest musk deer are high in captivity [8], and Eimeria sp. infection may be one of the reasons. In general, more pathogenic species tend to inhabit the posterior part of the intestines [30]. Coccidia can invade and destroy intestinal epithelial cells of the host, resulting in digestive dysfunction, acute or chronic damage, and induce secondary damage, the above symptoms are more obvious in young animals [31,32]. These are extremely unfavorable to the development of forest musk deer breeding industry.
In China, molecular data on Eimeria sp. in forest musk deer were not available. In our study, the infection level of intestinal parasites in captive forest musk deer was relatively high, which may be due to the single captive breeding mode in 7 regions. Because the captive mode of forest musk deer mainly consists of several individual brick cells and an outdoor yard (activity site), generally 2-6 single brick cells supported a large enclosure (activity site). Transmission is most e cient on farms with high stocking densities [22]. Parasites seemed to spread in forest musk deer in the same outdoor yard, which may become chronic carriers. Infected animals without clinical symptoms remain infected throughout the year and contaminate the environment continuously with oocysts [17]. Therefore, the infected forest musk deer are the source of reinfection and new infection in other animals. Oocysts can also be introduced into a susceptible herd by the fecal-oral route, contaminated clothing, boots, or cleaning tools [33,34]. At the same time, the arti cial enclosure can't completely simulate the eld environment, habitat changes make the animal in the state of stress for a long time, which will weaken their immunity [35], and then increase the parasitic infection rate.
The positive rates of Eimeria sp. infection was different in each areas, that may be related to the environmental climate, altitude, breeding and management mode of farms, and the control measures of parasites in various regions. Ma'erkang had the highest infection rate, which may be related to its large number of breeding and relatively old houses. The dissemination of coccidia is facilitated by high population density feeding [22]. The lowest infection rate was found in Hanyuan and Weinan, which might be the dosage and frequency of drugs used in Hanyuan and the management of the breeding farms in Weinan.
Regional factors seem to have an impact on the epidemiology of parasitic diseases, which can be attributed to geographical location and climatic conditions [36,37]. The altitude of Maoxian, Lixian and Ma'erkang was higher than that of other areas. The conditions of moderate heat and moisture in these areas are very favorable for the development, survival and transmission of Eimeria sp. [38]. Because for the reason that the oocysts can survive for weeks or months in such an environment [33], the positive rate in these areas is higher.
In addition, it is worth noting that the feeds provided to forest musk deer in different regions are not identical, which may make the composition of the intestinal microbial community of animals very different, thereby affecting the parasitism of coccidia in the intestine [39,40].
There is an increasing recognition that Eimeria sp. prevalence and intensity has a relevant relationship with seasonality [41]. Temperature, humidity, and rainfall are the main environmental factors affecting the survival and transmission of gastrointestinal parasites [34]. Eimeria sp. can remain viable and infectious for at least 1 year in vitro and able to withstand many adverse environmental in uences because of their thick oocyst wall [42]. However, direct exposure to ultraviolet light for a few hours or extremely dry is detrimental to the oocysts [43], none of the 7 areas we sampled had such climatic conditions. Sichuan and Shaanxi are similar in geographical location and climate. The annual average temperature is above 10 ℃, and the temperature uctuates greatly in different seasons. The precipitation in spring is several times more than that in winter. The total sunshine hours of the whole year are between 1500 ~ 2800 h [44,45]. In spring, oocysts tend to sporulate faster and disperse better with increasing precipitation [46], whereas cold and dry weather during winter inhibits development and transmission [34]. Therefore, the intestinal Eimeria sp. infection was more serious in spring. This situation should lead to the importance of farming work and strengthen the seasonal prevention and control of coccidiosis.
In this study, the 18S RNA gene sequence of different species of Eimeria sp. was highly similar. These results indicated that the 18S rRNA gene sequences of Eimeria sp. parasitized in captive forest musk deer in this study were homologous between species.
The 18S rRNA gene sequences of 34 types of Eimeria sp. determined in 7 regions had some base variations, and the similarity between sequences was 90.5% ~ 100%, which may be due to the fact that ribosomal rRNA gene was a highly repetitive tandem sequence unit in eukaryotes and its 18S rRNA gene sequence was highly conservative in the evolution process of biological genetic population [28,47,48]. Eimeria sp. has a wide variety of species and can be parasitic on many animals, such as chickens, rabbits, cattle, sheep, goats, and deer, etc. Eimeria sp. is considered to have high host speci city and has strict selectivity to parasitic host animals and parts [18]. Therefore, each host animal has its own Eimeria origin in the classi cation of Eimeria sp. and cannot cross-infect each other [19,49,50].
According to the phylogenetic tree, DJY3 and Eimeria ahsata were in the same branch. This may be related to the fact that sheep, cattle and musk deer were ruminants. Combining with the phylogenetic tree, it showed that the closer the relationship of host animals was, the closer the relationship of Eimeria sp. parasitized on host animals was [51,52].

Conclusions
This experiment con rmed the prevalence of intestinal coccidiosis in captive forest musk deer in China.
As far as we know, this is the rst molecular identi cation of Eimeria sp. in the intestinal tract of forest musk deer. This study showed that there were signi cant differences in the prevalence of coccidiosis in different regions, altitudes and seasons. Based on the sequence identi cation of 18S rRNA gene, it was con rmed that Eimeria sp. parasitized in the intestinal tract of forest musk deer was closely related to Eimeria alabamensis from Bos taurus, Eimeria faurei from Ovis aries and Eimeria ahsata from Ovis aries. At present, there is no known effective drug to treat the Eimeria sp. infection. Therefore, measures should be taken to prevent forest musk deer from being infected.

Declarations
Ethics approval and consent to participate In this study, the process of specimens collected was accorded with animal protection law of the People's Republic of China. And all institutional and national guidelines for the care and use of laboratory animals were followed and animal experiments were approved by the National Institute of Animal Health Care and Use Committee at Sichuan Agricultural University (approval number SYXK2019-187).

Consent for publication
Not applicable.

Availability of data and materials
All data involved and arising from the study are included in this published article.

Competing interests
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.    Sequence similarity analysis of Eimeria sp. 18S rRNA gene.