Zoonotic Trematode Prevalence In Galba Pervia (Lymnaeidae) And Experimental Infection Of Three Isolated Trematodes In The Intestine Of Duck

Jian Li Guangxi Traditional Chinese Medical University Yijing Ren Guangxi University Lei Yang Guangxi University Jiani Guo Guangxi University Haiying Chen Inner Mongolia University Jiani Liu Inner Mongolia University Haoqiang Tian Inner Mongolia University Qingan Zhou Guangxi University Weiyi Huang Guangxi University Wei Hu Inner Mongolia University Xinyu Feng (  fengxinyu2013@163.com ) Inner Mongolia University https://orcid.org/0000-0002-4120-7759

Galba pervia belongs to Mollusca, Gastropoda, Pulmonata, Basommatophora, Lymnaeidae, Galba [10]. It is an intermediate host for a variety of trematodes, some of which are zoonotic, such as F. gigantica, F. hepatica, Echinostoma revolutum, Echinochasmus perfoliatus and plays a vital role in the transmission and prevalence of these diseases [11]. The shell of G. pervia is thin and translucent with an ear-shaped aperture; the ratio apex/body is 10/8mm. Its natural habitats ranged from lakes, canals, ponds, and rice elds. Oviparous hermaphroditic G. pervia lives in large aggregation in suitable environments such as sewage sludge bottom or broken bricks and feeds on algae, hummus, and aquatic plants [12]. G. pervia is widely distributed in China and is the dominant host snail for transmitting Fasciola spp [13].
Food-borne trematode is often infected by eating raw vegetables such as sh mint (Houttuynia cordata), lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium o cinale) [14]. From 2011 to 2012, there was an outbreak of F. gigantica infection in Binchuan County, Dali Prefecture, Yunnan Province in China, and then the authors think that sh mint was most likely the source of diseases [9,15]. Guangxi Zhuang Autonomous Region is contiguous to Yunnan and shares a similar climate, as well as lifestyles and dietary habits of the local people. Given that the Galba pervia is the important intermediate host of Fasciola in China [11,16], it also has a wide distribution in Guangxi, representing a potential risk of parasitic zoonosis. Therefore, the main objective of this study was to investigate and identify presence of various trematode larvae in G. pervia in Guangxi, and assess the zoonotic potential of trematode for both animal and human in this area.

Study areas and snail collection
To investigate the potential vector capacity of G. pervia in Guangxi Province, snails were collected from 54 sites in 9 cities, namely, Beihai, Fangchenggang, Guigang, Guilin, Liuzhou, Laibin, Nanning, Qinzhou, Wuzhou, and Yulin, from 2012 August to 2014 August (the number of snail samples per site was about 200). Two types of areas were included: Type 1 areas were rice cultivation areas (contains 51 sites, marked by circular shapes in Fig. 1G); Type 2 areas were the vegetation areas of crops which often used as the raw food (10 sites, marked by triangular shape in Fig. 1G). Details of each locality sampled are given in Table S1. In each sampling site, the snails were collected manually by the plastic scoop, transported to the laboratory, cleaned and rinsed ve times in sterilized water, and then placed in plastic trays for subsequent experiments. Identi cation of snails and isolation of trematodes larvae The snails were identi ed morphologically as G. pervia depending on systematic keys of the shell [12]. Then collected G. pervia were dissected under a stereomicroscope and carefully checked for trematodes larvae (rediae, cercariae, or metacercariae), and the larvae were separated from the tissue. We used MoticBA400 microscope to observe and record the body length and body width of each isolated trematode. The body length and body width of rediae, and the body length, body width, tail long and tail width of cercariae at each site were measured. The diameter and wall thickness of metacercaria were also measured.

Molecular examinations of the trematodes
Next, a single larva with the identical morphology at each sampling site was selected and rinsed with sterilized distilled water three times before being used to extract parasite genomic DNA by a DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA samples were stored at −20°C until PCR ampli cation. The PCR assay targeting the sequence of the internal transcribed spacer 2 (ITS2) gene was used to amplify trematode larvae. The universal primer pairs were designed as described by McManus et al. [17]. All the PCR products were directly sequenced after being puri ed. The obtained sequences were edited using DNASTAR software (www.dnastar.com/software/lasergene/) and aligned using ClustalX (http://www.clustal.org/clustal2/). The identity of individual specimens was ascertained by comparison with the sequences available in 'non-redundant' database in GenBank by BLAST (http://www.ncbi.nlm.nih.gov/blast/). The nucleotide sequences obtained in the present study have been deposited in the GenBank database under the accession numbers. Cyathocotyle prussica (MH521249), the Brachylaima sp. (JX010634) and Schistosoma japonicum (S72866) were used as out-groups. The phylogeny was tested with 1,000 bootstrap replicates, using the Kimura two-parameter model as a nucleotide substitution model and gamma distribution as rates among sites.

Experimental infections of isolated trematodes in the intestine of duck
Five-day-old ducklings were fed with snails parasitized by isolated trematodes in the eld. Each duckling was fed 20 G. pervia, and one duckling was dissected every day from the 1st to 10th day after ingestion. The trematodes were collected from the duck intestines using a complete helminthological dissection method [5], and high-resolution pictures of the collected trematodes were taken with the Motic BA400 microscope and additional accessories. The carmine staining of the press-and-xed specimen was made according to the method provided by Kong Fanyao [6], and collar, spines, oral sucker, acetabulum, prepharynx, esophagus, testis, ovary was measured from digital images during daily observations. In addition, a single trematode was selected, and a small amount of tissue from the tail of the parasite was cut out aseptically. After repeated rinsing with sterilized distilled water 2-3 times, DNA extraction was carried out according to the above method. ITS2 gene was ampli ed and sequenced using the same method, and the trematode species was veri ed.

Results
Overall information on the sampling and survey data G. pervia samples were collected from 54 sites (as shown in Fig. 1G) with about 38-214 snails in each site. Trematodes were found in 17 sites investigated following dissection, including Tianbao Reservoir and Hede village in Nanning city, Liushan Town, Liutang Village, Guangrong village, and Cha Village in Liuzhou City, and Maling Town in Guilin City. Various stages in the life history of this trematode (redia, cercaria and metacercariae) were found during dissection procedures.

Morphological characters and molecular identi cation of trematodes larvae
The rediae of echinostomes were cylindrical, blunt at both ends, slightly pointed at the head and more pointed at the tail. The body was curved to the ventral surface with muscular feet, and the movement was slow. The tail of the cercariae is not forked. The head of H. conoideum cercariae shows prominent spines, as well as well-developed ventral suckers, pharynx, and intestines ( Fig. 2D-F). The metacercaria were round and have two transparent walls (the outer wall was thicker than the inner wall). Abdominal suckers and refractive granules of larvae could be seen inside the cyst. Due to the movement of the larvae inside the sac, the small spines around its head were not easily observed. The rediae of Australapatemon sp. forms a distinct bulge at the head. The cercaria larvae had a forked tail, which was obviously longer than the body length. The cercaria of P. cordatum also had visible forked-tail, oral sucker and pharynx (  The prevalence of trematodes in G. pervia The overall trematodes infection rate was 22.0% (1818/8258). Echinostoma revolutum were detected in the snails from 11 sampling sites, with an infection rate of 12.9% (1069/8258); Hypoderaum conoideum infection was detected in the snails from two sampling sites, with an average infection rate of 3.8% (315/8258). Infection of Australapatemon sp. was detected in the snails form 2 sampling sites, with an infection rate of 2.5% (206/8258); Infection of Pharyngostomum cordatum and Echinostoma sp. were detected at 1 sampling site with an infection rate of 0.4% (34/112) and 2.3% (194/8258), respectively.

Phylogenetic analyses
In total, 15 representative high-quality ITS2 sequence data was obtained. Figure 2 shows an NJ tree based on the submitted sequences and relevant GenBank sequences. The ITS2 sequences of Echinostoma sp. constituted a monophyletic clade (Fig. 3A shaded pink area), distinct from the clade formed by E. robustum, E. friedi and E. miyagawai. The sequences of E. revolutum and H. conoideum constituted a monophyletic group together with E. revolutum (AY168930) and H. conoideum (AJ564385) references (Fig. 3A shaded blue area). The ITS2 sequences of Australapatemon sp. formed a group with A. burti (KU950451) at 99% bootstrap value but formed a unique clade at 75% bootstrap value. Figure 3B showed that the ITS2 sequences of P. cordatum were identical to the reference sequences of P. cordatum (KJ137231).

Figure 3
Laboratory infection experiment with Echinostoma sp., E. revolutum, and H. conoideum Because there was no suitable second intermediate host for P. cordatum and de nitive host for Australapatemon sp., we conducted an infection experiment for isolated three kinds of trematode to evaluate rates of parasite establishment in ducklings. Ducklings were individually exposed to Echinostoma sp., E. revolutum, and H. conoideum larvae and all were successfully infected. Subsequent observation on Ducklings (17 dpi) fed with Echinostoma sp. infected G. pervia, we detected eggs (195.8×143.8 µm) in the feces, and the morphological characteristics of adult Echinostoma sp. were presented as measures: body length 9.8 mm, width 1.2 mm, oral suker 638.9×399.2 µm, acetabulum 1591.2×1338.2 µm, pharynx 492.6×331.7 µm, anterior testis 1120.5×707.4 µm, posterior testis 1274.9×880.4 µm, ovary 818.9×527.9 µm. In contrast, we found H. conoideum eggs in ducklings fed with infected G. pervia from three sites from a median of 12 dpi (range: 9 dpi to 14 dpi). The morphological characteristics of adult H. conoideum were: body length 1.05 mm, width 1. The developmental characteristics of E. revolutum in duckling host from juvenile to adult As there were not su cient metacercariae of other trematodes, experiments were only designed to gain insight into how E. revolutum developed in duckling hosts. The developmental characteristics of E. revolutum was recorded by dissecting infected ducklings from 1 dpi to 10 dpi (eggs in the feces were rst detected). E. revolutum could be obtained in the small intestine from 1 to 7 dpi and then migrate and reside in the cecum and colon around 8-10 dpi. The body length developed from 490 µm to 8500.5 µm (a dramatic 17-fold increase). At 1 dpi, juveniles presented a circumoral collar bearing 37 spines in a double circle and characterized by clearly visible oral suckers, acetabulum, pharynx, esophagus, and cecum. At 1 dpi, the tiny structure of the testis appeared. By 4 dpi, the ovaries were beginning to organize and develop, and the seminal receptacle began to form. The tubular-shaped uterus loomed at 4 dpi, and maturation of the reproductive and digestive organs occurred around 6~8 dpi. The vitelline glands were the last to appear, and several eggs deposited in the uterus could be observed at 9 dpi. E. revolutum larvae matured at 10 dpi and excreted eggs (Fig. 4). The daily measurement of E. revolutum development was recorded in detail, as shown in Table S2. Discussion Numerous species of food-borne trematodes are endemic in developing nations and signi cantly impact public health [18,19,20]. Austropeplea, Galba, Lymnaea, Radix and Stagnicola etc. from the families Lymnaeidae act as intermediate hosts of trematodes with substantial implications for human health [10,13]. The primary research focused on the capability of transmitting Fasciola sp., and at least 20 species of Lymnaeidae have been described as potential vectors of fascioliasis [21]. The results reported in the present paper demonstrate the presence of ve trematode species in G. pervia. Morphological characteristics identi ed the larvae to species level by combining unequivocal molecular markers, which identi ed as E. revolutum; H. conoideum; Australapatemon sp. P. cordatum and Echinostoma sp., respectively. Different collection sites differed concerning the larvae species and intensity of snails present, which would link with meteorological parameters and habitat types.
However, other trematode fauna, such as Fasciola, has not been detected, although Guangxi is one of the important regions of ruminant fascioliasis prevalence in the previous reports [22]. Our investigation indicated that E. revolutum was the most prevalent trematode species in Guangxi Province, with an infection rate of 12.9% among collected snails. In consideration of previous studies that Echinostomatidae have low intermediate host speci city [23]. In addition, Radix plicatula, R. swihoei, Gyraulus conrexiusculus etc. can also act as intermediate hosts [24], and all of above-mentioned snail species also have a wide distribution in Guangxi Province, so it implicates that the actual infection rate of Echinostomatidae trematodes may be much higher than the results found in this study.
There are many species of echinostomes, which are tiny parasites that mainly parasitize the intestines of birds, mammals, and humans [21,25,26]. However, due to the high diversity of species and similar morphology, some species have not been fully morphologically described by the most used morphological traits, with a precise classi cation elusive. In addition, it is time-consuming to identify the adults by reintroducing the larvae to complete their life cycles, and the improper selection of the de nitive experimental host will also lead to the failure of entering the next stage of the life cycle. Given these facts, Jonsson et al. proposed to apply gene markers or restriction fragment length polymorphism (RFLP) for molecular identi cation [17,21,27,28]. ITS2 species-speci c markers have been proven as suitable genetic markers for identifying and differentiating trematode species. Because the external morphology of trematode metacercariae from this study was quite similar, the ITS2 gene sequences of metacercariae were ampli ed to identify the metacercariae. The species identi cation results are consistent with morphological analyses, and the evolutionary relationships of trematode species were successfully elucidated and compared with reference sequences deposited in the public databases.
As early as 1968, Lie [29] et al. proposed that the development of trematodes may be restricted by others due to the competition inside the snail when they take the same species of snail as the intermediate host.
Subsequent studies revealed a similar competition relationship in the intermediate host of echinostomes [30] and schistosomes [31]. In our study, different trematode species have not been detected in one snail simultaneously. Meanwhile, although G. pervia snail can also serve as the intermediate host for Fasciola in Guangxi Province, we have not observed Fasciola infected G. pervia. This phenomenon may be caused by the cross-species competitive antagonisms of echinostomes with other trematodes, which led to a generally low or non-infection of Fasciola. Trematode is a parasite that can cause severe zoonotic diseases. Lie proposed that the transmission of the disease could be contaminated through competition among trematode larvae in intermediate hosts in 1973 [32]. Although theoretically, echinostomes could be used to reduce the economic losses caused by Fasciola, however, given its great harm to the livestock and poultry, echinostomes are not sound biological control agents for the control of Fasciola in practice. To unveil the trematode infection rate in larger areas in Guangxi Province, it is necessary to expand the sampling sites and select more species of snails for investigation. Further research is needed to determine the coexistence and coevolution of competitive species, especially two or more trematodes that reside in one snail host in natural communities.
Echinostomes are a common intestinal parasitic trematode in poultry, which mainly affects the growth and development of the young while is less harmful to the adult. The developmental cycle of echinostomes in its terminal host is short and uncomplicated. Therefore, the animal developmental model of the echinostomes in its terminal host is suitable for studying the immune response between trematode and its host. The research results can also be used as a reference for other small intestinal ukes which induce the terminal host immune response, and related research has also been reported in recent years [3][4]. This study is mainly aimed at the observation of the growth and development of echinostomes from decapsulation of the cyst to the sexually mature adult stage in the intestinal tract of the terminal host. For the selection of experimental animals, mammals are not susceptible to echinostomes infection, so ducklings were used as the de nitive host in our study. To provide a basis for subsequent related research, it needs to explore more animal models for echinostomes infection in the future.

Conclusions
Page 10/14 The present investigation revealed the prevalence of ve trematodes species in the G. pervia in Guangxi Province, China. The results from our study not only provide a baseline information but also offered laboratory experimental models for assessing the potential zoonotic echinostomiasis from G. pervia. Further research is needed toward the understanding risk of human infection in combination with risk evaluation to ameliorate unwanted adverse effect during casual contact or exposure to infected G. pervia.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
All participants consented to have their data published.

Availability of data and materials
The sequences data has already submitted to GenBank, and will be released to the public database until Dec 1, 2016. The GenBank accession numbers are KX781395 for the ITS2 of Australapatemon sp., KM980463~KM980465, KM980478~KM980479 for the Hypoderaeum conoideum, KM980474 and KM980476~KM980477 for the Echinostoma revolutum.

Competing interests
The authors declare that they have no competing interests.

Authors' Contributors
LJ and RYJ conceived and designed the study. LJ, RYJ, LY, GJN and CHY, LJN, THQ, ZQA and HWY collected and identi ed the snails, cercariae and metacercariae. RYJ and LJ analyzed the data and drafted the manuscript. LJ, FXY and HW helped in study design, study implementation and manuscript revision. LJ, FXY and HW critically revised the manuscript. All authors read and approved the nal manuscript.