First detection of Blastocystis sp. in migratory whooper swans (Cygnus cygnus) in China

Abstract

Blastocystis is a usual intestinal protist that always found in humans and various animals. Currently, the prevalence of Blastocystis in the migratory whooper swan (Cygnus cygnus) is unknown. In our research, we aimed to determine the occurrence, prevalence, subtype distribution and dynamic transmission mechanisms of Blastocystis in the migratory whooper swan in China. We also assessed the zoonotic potential of Blastocystis isolates, as well as possible routes of transmission and impact of this organism on One Health perspective. Fecal samples (n = 770) were collected from whooper swans inhabiting the Sanmenxia Swan Lake National Urban Wetland Park, China. The overall prevalence of Blastocystis was 11.6% (89/770). We identified 9 subtypes of Blastocystis sp., including 5 zoonotic subtypes [ST1 (Cakir et al., 2019 (8)), ST4 (Selma and Karanis, 2011 (4)), ST5 (Stensvold et al., 2009 (1)), ST6 (Fare et al., 2019 (5)) and ST7(58)] and 3 host-specific subtypes [ST10 (Zhao et al., 2018 (7)), ST14 (Tan et al., 2010 (2)), ST23 (Wang et al., 2018 (3)), and ST25 (Stensvold et al., 2009 (1))]. Subtypes ST4, ST5, ST6, ST10, ST14, ST23, and ST25 were first identified in the whooper swan. Among these subtypes, ST23 and ST25 were identified in birds for the first time, indicating that these subtypes are expanding their host range. So far, this is the first research reporting on the prevalence and subtypes distribution of Blastocystis in the migratory whooper swan in China. The findings obtained in this study will provide new insights into the genetic diversity and transmission routes of Blastocystis, and the possible public health concerns posed by this organism.

Highlights

• •ST1, ST4, ST5, ST6 and ST7 were identified in whooper swans in China.
• •ST4, ST5, ST6, ST10, ST14, ST23 and ST25 were first identified in whooper swan, ST23 and ST25 were identified for the first time in birds.
• •Public health and ecological significance of migratory birds-borne Blastocystis sp. at “One Health” perspective.

1. Introduction

Blastocystis sp. are anaerobic enteric protozoans commonly detected in the gastrointestinal tracts of humans and a wide range of animals, including asperissodactyls, artiodactyls, rodents, proboscideans, marsupials, non–human primates (NHPs), reptiles, birds, annelids, insects, amphibians, and fish [[1], [2], [3]]. Blastocystis cysts are transmitted to humans mainly by the way of ingestion of contaminated water or food, exposure to fecal contamination, and person–to–person contact [4,5]. The clinical manifestations caused by Blastocystis are vary from self–limiting abdominal discomfort to chronic persistent diarrhea and skin lesions [6,7]. Clinical symptoms and severity of disease depend on different Blastocystis subtypes. A recent study has shown that Blastocystis is more usual detected in healthy individuals than in those with diarrhea or other gastrointestinal symptoms; Blastocystis is also found in young and immunocompromised individuals [8].

Extensive genetic diversity within the Blastocystis have been revealed by PCR–based analyses among epidemiological studies. Among the 22 published Blastocystis subtypes (STs 1–17, ST21, and STs 23–26), ST1-ST9 and ST12 have been always detected in humans, with ST1–ST4 more common [9,10]. ST5 has been found in Perissodactyla and Artiodactyla, ST6 and ST7 have been found in birds, and ST8 has been found in non–human primates (NHPs) [11]. However, ST10 and ST14 are only characterized in specific animal, suggesting host specificity [12]. Several studies have shown that mixed subtypes occur in a variety of animals [11,13,14]. Evolutionary sources of Blastocystis genus various are mainly from inter–subtype recombinant and transmission of infection between host species [15,16].

Numerous surveys about the prevalence and subtype distribution of Blastocystis sp. in various animal have been reported [[1], [2], [3]]. However, whooper swan (Cygnus cygnus) has not been investigated, and available data are scarce. Whooper swan is flock–dwelling herbivorous migratory waterfowl, which is migrate timed and directionally, and stop in specific areas [17]. Indeed, migratory whooper swan have long migration paths and can travel through cities, freshwater lakes, low–lying coastal agricultural lands, wet pastures, and forests, stopping to rest in areas that contain bodies of water. Because of the presence of avian influenza (H5N1) and zoonotic intestinal parasites (Cryptosporidium spp. and Enterocytozoon bieneusi) in migratory whooper swans, so increased concern and worry of humans about the risk of diseases spreading [[18], [19], [20], [21]]. Some studies have shown that whooper swans carry viruses for long–distance transmission, and that the continuous evolution of viruses may be related to the routes of migratory birds [22,23].

Every winter, >10,000 migratory whooper swans stay in Sanmenxia Swan Lake National Urban Wetland Park for spend the winter [24]. However, humans feeding has led to a dramatic increase in the population of whooper swans (Fig. 1). The main migration route of the whooper swan is from Sanmenxia to Yumenkou wetland, then into the Yellow River of Mongolia, and finally to the central and western parts of Mongolia. However, whooper swan at least rest on 40 watersheds and lakes among migration, thus posing a potential threat to public health [25]. In order to protect the health of the whooper swan and avoid potential public health risks, it is necessary to enquire the occurrence of Blastocystis in the whooper swan. Therefore, the purpose of present study is to know and confirm the infection status of Blastocystis in migratory whooper swan in China. And the possible route of transmission and the significance of Blastocystis on public health is also discussed.

Fig. 1.

Migratory whooper swans (Cygnus cygnus) at the Swan Wetland Park in Sanmenxia, China.

A: Migratory whooper swan in flight at the Swan Wetland Park in Sanmenxia, China; B: Migrating whooper swans in water and wetlands at the Swan Wetland Park in Sanmenxia, China.

2. Materials and methods

2.1. Sample collection

A total of 770 fresh fecal samples were collected from the whooper swans inhabiting the Sanmenxia Swan Lake National Urban Wetland Park in the city of Sanmenxia, located in the middle reaches of Yellow River, China (Fig. 1). Research shows that wintering period includes early, middle and late: October–November (arrival) is the early wintering period, December–January of the following year is the middle wintering period (this period shows the largest number of birds and most stable community structure), and February–March is the late wintering period (during which the birds leave after the completion of their pre–breeding molt) [25]. So that understand the dynamic transmission mechanism of Blastocystis in the migratory whooper swan, we collected fresh fecal samples four times according to the wintering period (Table 1). For each sample, in order to avoid environmental pollution, only the central portion of the fresh fecal was collected into a sterile glove, keep in ice box, and sent to our laboratory, stored at 4 °C.

Table 1.

Prevalence and subtypes of Blastocystis sp. in whooper swans (Cygnus cygnus) in Sanmenxia, China.

Collection date	Whole winter	Positive/Total samples (%)	Subtype (no.)
2018.11.17	Early-term	13.1% (31/237)	ST1 (7); ST6 (1); ST7 (17); ST10 (3); ST14 (1); ST23 (1); ST25 (1);
2018.12.06	Mid-term	11.2% (18/161)	ST7 (15); ST6 (3)
2019.03.18	Late-term	8.7% (6/69)	ST6 (1); ST4 (2); ST7 (3);
2019.12.09	Mid-term	11.1% (34/305)	ST1 (1); ST4 (2); ST5 (1); ST7 (23); ST10 (4); ST14 (1); ST23 (2);
Total		11.6% (89/770)	ST1 (8); ST4 (4); ST5 (1); ST6 (5); ST7(58); ST10 (7); ST14 (2); ST23 (3); ST25 (1)

2.2. DNA extraction

For each sample, fecal whole genomic DNA was extracted based on the instructions of E.Z.N.A.R Stool DNA Kit (Omega Bio–Tek Inc., Norcross, GA, USA). The quality of the DNA samples was assessed using Nanodrop One, and high–quality extracted samples were then labeled, sub–packaged into 200 μl sterile centrifuge tubes, and stored at −20 °C.

2.3. PCR amplification

Blastocystis sp. in all samples were detected using nested PCR amplification of the partial small subunit (SSU) rRNA gene. The external primers and internal primers as previously described, which produced a PCR product size of 479 bp [26,27]. PCR assays included positive and negative controls and all samples were tested in triplicate. The PCR products were mixed with DNA Green reagent (Tiandz, Inc., Beijing, China) for staining (1:4), and then detected by 1.0% agarose gel electrophoresis for the presence of the target fragment in the PCR amplification products. The PCR amplification products and positive control samples with the presence of the target fragment were sent to Sangon Biotech (Zhengzhou, China) first–generation bidirectional sequencing.

2.4. Sequencing and phylogenetic analysis

After all PCR amplification products and positive control samples with the presence of the target fragment were processed according to the instructions of the Cycle Sequencing Kit (BigDye Terminator v3.1), they were sequenced using the 3730xl DNA Analyzer (ABI PRISMTM). The sequence of the PCR product sample was detected by NCBI Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov) and identified by alignment with a highly homologous reference sequence to identify Blastocystis sp. Edited the sequence in Clustal X 2.1 software (http://www.clustal.org/) according to the reference sequence with high homology. The phylogenetic evolutionary tree of Blastocystis sp. was constructed and analyzed using MEGA 7.0 software (Kimura two–parameter model, neighbor–joining algorithm) (http://www.megasoftware.net/).

2.5. Statistical analysis

Differences in infection rates with Blastocystis sp. between samples collected at different times were analyzed using 95% confidence intervals (CI) and the chi–squared test, which were bast on the SPSS software 22.0 version (SPSS Inc., United States). Significant differences were considered when P < 0.05.

3. Results

3.1. Blastocystis prevalence

The overall prevalence in 770 fecal samples collected from whooper swans was 11.6% (89/770). The prevalence differed according to different sampling times. Early–term showed the highest infection rate at 13.1% (31/237), while late–term showed the lowest at 8.7% (6/69). Significant differences (P > 0.05) in Blastocystis prevalence among the four times of collection, ranging from 8.7% to 13.1% (Table 1).

3.2. Distribution of Blastocystis subtypes

A total of nine Blastocystis subtypes were found: ST1 (n = 8), ST4 (n = 4), ST5 (n = 1), ST6 (n = 5), ST7 (n = 58), ST10 (n = 7), ST14 (n = 2), ST23 (n = 3), and ST25 (n = 1). Among these subtypes, ST1, ST4, ST5, ST6, and ST7 were zoonotic subtypes (Table 1), 7 (ST1, ST6, ST7, ST10, ST14, ST23, and ST25) were found in the 31 Blastocystis samples from early–term collection time, 8 (ST1, ST4, ST5, ST6, ST7, ST10, ST14, and ST23) were detected in samples collected during middle–term, and 3 (ST4, ST6, and ST7) were detected in samples collected during late–term. ST25 and ST5 were detected only in the early and mid–collection samples, respectively. ST7 was the predominant subtype, and no mixed subtypes infection was found in present research.

3.3. Phylogenetic analysis of Blastocystis sp.

In our study, we sequenced 89 positive isolates and obtained 13 representative sequences. The sequences obtained are highly homologous to the reference sequence of Blastocystis sp. in the GenBank database. (Fig. 2). Eight ST1 isolates produced two variations, and ST1A was the predominant subtype. ST1A was detected in early–term and ST1B was detected in mid–term. Two variations of subtype ST7 were found and subtype ST7A (n = 47) was the dominant subtype. ST7A and ST7B were both detected in three periods of winter. Two variations were identified in ST10, and subtype ST10A (n = 4) was the predominant subtype. ST10A was detected in mid–term and ST10B was detected in early–term. Sequence of the ST10A isolate was consistent with that detected in cattle in the USA; the GenBank sequence accession number for this sequence was MK244921. Two ST14 isolates produced two variations: ST14A was detected in early–term and ST14B was detected in mid–term. Phylogenetic analysis revealed more clearly the polymorphisms in the Blastocystis isolates detected in present study.

Fig. 2.

Neighbor-joining tree of Blastocystis subtypes isolated from the whooper swan, based on SSU rRNA sequences.

Phylogenetic relationships among the 18S nucleotide sequences of Blastocystis subtypes examined in our study, and among other reported Blastocystis subtypes. Phylogeny was inferred using the neighbor–joining method. Bootstrap values were obtained using 1000 replicates; those with >50% support are shown on the nodes. The Blastocystis sp. identified in this study are designated using filled triangles.

4. Discussion

So far, reports of Blastocystis in swan were limited to small population of swan in zoos or parks. Details are as follows: swan goose (Anser cygnoides) in Brazil (100%, 1/1), swan in Malaysia (35%, 7/20), swan goose (Anser cygnoides) in Spain (16.6%, 3/18), and black swan (Cygnus atratus) in China (10.5%, 4/38) [[28], [29], [30], [31]]. It should be noted that all of the previous studies were conducted using small sample sizes. However, several studies have reported on Blastocystis in captive and wild birds. Indeed, Blastocystis has been detected in birds (wild and domestic) around the worldwide, with the infection rate ranging from 2.1 to 100% [32]. In our present study, Blastocystis prevalence was 11.6% in the whopper swan inhabiting the Sanmenxia Swan Lake National Urban Wetland Park, China. This percentage is within the range reported in other studies conducted in birds, and is lower than the 90% reported in wild bird species in Colombia, 23.4% in bird species (farm, bush land, and zoo) in Australia, and 21% in captive and wild bird species in Brazil [31,33,34]. However, the prevalence observed in present research was higher than those in captive wild birds in China (10.5%) and those in bird species in French zoos (8.6%) [30,35]. These previous studies also show that the infection rate was varies widely, so more studies are needed to decide what factors might influence this variability.

A total of fourteen subtypes (ST1, ST2, ST4, ST5, ST6, ST7, ST8, ST10, ST13, ST14, ST20, ST24, ST27, and ST28) of Blastocystis have been detected in birds around the worldwide [32]. In our study, ST1, ST4, ST5, ST6, ST7, ST10, ST14, ST23, and ST25 were detected in the whooper swan. To the best of our knowledge, only ST1, ST3, ST7, and ST8 have been found in swan worldwide [[28], [29], [30], [31]]. Therefore, this is the first research to report on the occurrence of subtypes ST4, ST5, ST6, ST10, ST14, ST23, and ST25 in the swan. ST7 and ST1 were the first and second dominant subtypes found in swan, respectively. The presence of these two subtypes in the swan further confirms the suitability of the swan as host organism for ST7 and ST1. The frequent occurrence of subtypes ST1, ST6, ST7, and ST10 in the swan evaluated in our present study further backs the notion that birds may be common hosts for these subtypes. Isolates ST1A, ST10B, and ST14A, and isolates ST1B, ST10A, and ST14B, were only detected in the early and mid–migration stages, respectively. Hence, migration may promote recombination or reassortment of Blastocystis genes, resulting in the generation of numerous new subtypes or isolates. The continuous circulation of Blastocystis with seasonal changes in the epidemic areas may also influence this process.

Some studies have shown that ST1 and ST4 are most common in human infection. ST1 and ST4 have also been detected in different animals (NHPs, livestock, birds, wildlife, companion animals, and marine mammals), sewage/wastewater, and surface and drinking water [[30], [31], [32], [33], [34], [35], [36]]. Zoonotic subtype ST5 has been found in ostriches, rodents, pigs and NHPs [4,31,32,35]. ST6 and ST7 are generally regarded to be bird subtypes, which were also occasionally found in human and certain mammals (NHPs, cattle, goats, pigs and dogs) [[6], [7], [8], [9],32,35,36]. Therefore, ST1, ST4, ST5, ST6, and ST7 pose potential risk for zoonotic transmission or for waterborne transmission. Four non–zoonotic subtypes (ST10, ST14, ST23, and ST25), which are commonly isolated in animals and always regarded animal–specific subtypes, are frequently detected from cattle, deer, yak, alpacas, and goats [[3], [4], [5], [6],32,[37], [38], [39]]. In our present study, subtypes ST23 and ST25 were found in birds (the whooper swan), indicating that these two subtypes are expanding their host range.

The data obtained thus far suggest that the whooper swan is a natural reservoir of Blastocystis, indicating that whooper swans can carry Blastocystis for long–distance transmission. The migratory whooper swans have long migration paths and can travel through cities, forests, wet pastures, low–lying coastal agricultural land, and freshwater lakes, stopping to rest in areas that contain bodies of water [17]. These findings indicate that the migration routes of the whooper swan play an important role in the geographic spread of Blastocystis. Blastocystis cysts can be transmitted to other birds (such as water, ground, and tree birds) and animals (wildlife, livestock, captive farm and companion animals, and marine mammals) via the fecal–oral transmission chain during migration. Additionally, Blastocystis is always found in water (such as sewage/wastewater, and surface and drinking water), on vegetables, and in air [36,40,41]. Migratory whooper swans prefer to live in water, and feed on grains and vegetables [17]. Previous studies have suggested that may be more susceptible to budding Blastocystis cyst infection due to ground and water birds feeding habits [42]. Roosting sites may serve as reservoirs of Blastocystis cysts. Blastocystis–infected whooper swans excrete fecal matter when roosting, and Blastocystis is then transmitted to susceptible birds via oral ingestion of contaminated water and food (Fig. 3).

Fig. 3.

Schematic diagram shows the ecological and public health significance of Blastocystis sp. in the migratory whooper swan and major routes of Blastocystis transmission.

Possible direct, indirect, waterborne, and foodborne transmission of Blastocystis sp. by the migratory whooper swan at the human–animal–environmental interface.

Humans can be directly and indirectly infected with Blastocystis cysts through contact with the migratory whooper swan. During the migration process, migratory whooper swans can stopover in cities (parks, lakes, and rivers) to feed, rest, and even settle in for the winter [17]. Humans feeding has led to a dramatic increase in the population of whooper swans. However, this behavior can lead to direct contact between humans and the migratory whooper swan, thereby increasing the chance of human contact with migratory whooper swan droppings. Migratory whooper swans also stop in areas containing bodies of water in order to rest and defecate in the water. This activity pollutes the water and can lead to Blastocystis transmission to humans through drinking water. When migratory whooper swans forage in low–lying agricultural land and vegetable fields, they can directly contaminate crops and vegetables. Polluted waste and surface water may contaminate food via the process of crop and vegetable irrigation, while fish obtained from polluted water can cause foodborne transmission of Blastocystis [43]. Waterborne and foodborne transmission of Blastocystis is a public health concern that should not be ignored (Fig. 3).

To date, multiple zoonotic potential of organisms has been reported in migrating swans. For example, avian influenza (H5N1), Cryptosporidium spp. E. bieneusi, Blastocystis sp. (in present study), Campylobacter spp., Streptococcus bovis, St. gallolyticus, St. pneumoniae, Enterococcus faecalis, En. faecium, Clostridium difficile, Clostridium botulinum, Clostridium tetani and Clostridium perfringens [[18], [19], [20], [21]]. As the causes of several diseases in humans and animals, the above organisms can persist in the environment for a long time, so they are always considered as indicators of water pollutant monitoring [[44], [45], [46]]. The above results indicate that migratory swans are a potential public health threat. Therefore, there is a need to strengthen the environmental health management of the breeding and wintering grounds of migratory swans to avoid their contamination of water sources and human infections.

5. Conclusions

The migratory whooper swan is a natural reservoir of Blastocystis. The nine Blastocystis subtypes detected in the migratory whooper swan in China include subtypes ST1 and ST4, which cause >95% of human blastocystosis. These data suggest that migratory whooper swans infected with Blastocystis show significant zoonotic potential. The migration process of the migratory whooper swan plays an important role in the geographic spread of Blastocystis by promoting the generation of new subtypes or isolates. Additionally, migratory whooper swans may constitute a direct or act as potential mediators, of Blastocystis transmission.

Funding

This work was partially supported by the National Key Research and Development Program of China (grant number 2019YFC1605700), the Leading Talents of Thousand Talents Program of Central China (grant number 19CZ0122), the National Natural Science Foundation of China (grant number 32102689), and the Outstanding Talents of Henan Agricultural University (grant number 30501055).

Ethical standards

This study was conducted according to the Regulations for the Implementation of the People's Republic of China on the Protection of Terrestrial Wildlife and Law of the People's Republic of China on the Protection of Wildlife. The research protocol used in this study was reviewed and approved by the Research Ethics Committee of Henan Agricultural University. During our study, no migratory whooper swans were harmed.

Author statement

Longxian Zhang contributed to the conceptualization and funding acquisition of the study. Kaihui Zhang, Ziyang Qin, and Huikai Qin contributed to the investigation, methodology data curation, formal analysis and writing-original draft writing of the study. Yinlin Wang, Luyang Wang, and Yin Fu contributed to the project administration, resources and software of the study. Changjiang Hou, Chenxiao Ji, and Yuan Yuan contributed to the supervision of the study. All of the authors have read and approved the final version of the manuscript.

Declaration of Competing Interest

The authors have no conflicts of interest to declare.

Data availability

All the data generated and analyzed in this study are included in the published manuscript. Nucleotide sequences for the SSU rRNA obtained in this study have been deposited into GenBank (under GenBank accession numbers ON394479–ON394487 and ON406354–ON406357).