EDP Sciences logo
Submit your paper
Search
Menu
All issues Volume 32 (2025) Parasite, 32 (2025) 23 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 23
Number of page(s) 10
DOI https://doi.org/10.1051/parasite/2025015
Published online 09 April 2025

© Y. Zhang et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

The Milu deer, or Père David’s deer, Elaphurus davidianus Milne-Edwards, 1866, the sole species within its genus, is classified under the order Artiodactyla, family Cervidae, and subfamily Cervinae [21, 47]. After a three-step strategy of population recovery, ex-situ conservation, and rewilding release, the population has grown to over 12,000 individuals across more than 90 populations [30]. The re-introduction and revitalization of the Milu deer is a global conservation triumph, demonstrating substantial success in conservation and reintroduction efforts, and exemplifying best practices in biodiversity conservation for United Nations Ecosystem Restoration projects [51]. Nevertheless, increasing population densities have exacerbated conflicts between the deer and their habitats, particularly as parasitic co-colonizations have contributed to a higher incidence of diseases, thereby limiting the healthy development of the individual [13, 23, 45, 48–50].

Trichomonads, a diverse group of anaerobic flagellated protozoa belonging to Parabasalia, are characterized by the presence of hydrogenosomes instead of mitochondria and the unique morphology of their flagella: flagellar apparatus, paraflagellates, and paraflagellar filaments [1, 7, 8]. Of the approximately 500 described Parabasalia species, the vast majority are endosymbionts, commensals, or parasites of various vertebrates (especially termites) and invertebrates, typically residing in the distal portion of the digestive tract [8]. Recently, six free-living species have been identified, mainly from freshwater and marine sediments [11]. Historically, the phylum parabasalia was divided into two groups based on morphological characteristics; however, the current taxonomic system recognizes six classes: Trichomonadea, Hypotrichomonadea, Tritrichomonadea, Cristamonadea, Spirotrichonymphea, and Trichonymphea [8, 9]. Although some species are harmless, several are pathogenic and of significant medical and veterinary importance [38], including Trichomonas vaginalis, which infects the genitourinary tract [28]; Trichomonas tenax, which is parasitic in the oral cavity [16]; and Pentatrichomonas hominis and Dientamoeba fragilis, which are present in the gastrointestinal tract and represent major human pathogens [26, 39]. Additionally, several species such as Trichomonas gallinae, Tetratrichomonas gallinarum, and Histomonas meleagridis, are avian pathogens [2, 5, 52], and Tritrichomonas foetus is known to cause abortion in pregnant cows [37].

Recent studies have demonstrated that trichomonads can parasitize non-unitary sites in hosts, adapt to new hosts, and pose a zoonotic risk [4, 6, 19, 27]. Consequently, there is growing interest in studying trichomonads to identify species that may exhibit zoonotic characteristics [10]. To date, little information exists on the occurrence and genetic diversity of trichomonads in Milu deer. Therefore, the present study aimed to determine the occurrence, risk factors, and genetic diversity of trichomonads in Milu deer, providing scientific guidance for ex-situ conservation of Milu deer.

Materials and methods

Ethical approval

This study adhered to the Guide for the Care and Use of Laboratory Animals set forth by the Ministry of Health, China. The protocol received approval from the Research Ethics Committee of the Anhui Agricultural University (number AHAUB2022019). Throughout the sample collection process, care was taken to ensure that the animals were not disturbed and stressed.

Study sites, sample collection, and sample processing

The present study was conducted at the following five sampling sites: (1) Beijing Nanhaizi Milu Park National Natural Reserve, located in the northern part of the North China Plain and the southern edge of the Yanshan Mountains; (2) Hebei Upstream of Luanhe River National Nature Reserve, located at the confluence of the Yinshan Mountains, the Daxingan Mountains and the remnants of the Yanshan Mountains; (3) Hubei Shishou Milu National Nature Reserve, located at the geographical center of the middle reaches of the Yangtze River; (4) Jiangsu Dafeng Milu National Nature Reserve, located on the coast of the Yellow Sea, east of Jiangsu Province, south of Yancheng City, and east of the Yellow Sea; and (5) Inner Mongolia Daqingshan National Nature Reserve, located in the Yinshan Mountains of the Inner Mongolia Autonomous Region. The data used to evaluate the possible risk factors associated with colonization were recorded, including the geographic coordinates, annual mean temperature, relative humidity, average annual rainfall, altitude, and number of individuals per hectare in these nature reserves (Table 1).

Table 1

Summary of characteristics of the five Milu nature reserves in China.

A total of 112 fecal samples were collected from the Milu deer National Nature Reserve from September 2022 to December 2023. These specimens were from five localities (Table 2), including Beijing Nanhaizi (n = 46), Hebei Upstream of Luanhe River (n = 13), Hubei Shishou (n = 12), Jiangsu Dafeng (n = 15), and Inner Mongolia Daqingshan (n = 26). Fresh and relatively intact feces were collected immediately after Milu deer defecation. To ensure standardization of the fecal sample collection process and to reduce potential contamination between samples, all fecal samples were collected with the assistance of experienced Nature Reserve staff. During sample collection, only the middle layer of feces was collected and stored separately in an insulated sampling box. All fecal samples were submitted to the laboratory under ambient storage and examined by light microscopy within 4 h. Following this, the remaining portions of all the samples were then immediately stored at −80 °C until DNA was extracted.

Table 2

Occurrence of trichomonad colonization in Milu deer. A potential risk factor for colonization was considered when p < 0.05 and OR > 1.

DNA extraction and PCR amplification

Genomic DNA was extracted from approximately 200 mg of each fecal sample using a TIANamp Stool DNA Kit (Beijing TransGen Biotech Co., Ltd., Beijing, China), according to the manufacturer’s instructions. Genomic DNA was eluted into 50 μL of elution buffer and stored at −40 °C until PCR amplification.

PCR was amplified, targeting an approximately 350 base pair (bp) fragment in the ITS-1/5.8S/ITS-2 sequence to determine the trichomonad species. The primers were TFR1 (5′-TGCTTCAGTTCAGCGGGTCTTCC-3′) and TFR2 (5′-CGGTAGGTGAACCTGCCGTTGG-3′) [11, 35].

The amplification process was performed in 25 μL volumes, including 1 μL of template DNA or primary PCR product, 2.5 μL 10 × KOD-Plus PCR buffer, 2.5 μL dNTPs (2 nM), 1.5 μL MgSO4 (25 nM), 0.5 μL of each primer (25 nM), 16 μL double-distilled water, and 0.5 μL KOD-Plus amplification enzyme (1 unit/μL) (ToYoBo Co., Ltd., Osaka, Japan). There was an initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 45 s, 55 °C for 45 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min in PCR amplification. The positive (DNA from the PCR-positive snake fecal samples) and negative (1 μL double-distilled water replaced 1 μL template DNA) controls were used in each PCR amplification. The reactions were performed in a thermocycler (Applied Biosystems, Foster City, CA, USA). The final products from the amplification were analyzed by electrophoresis on 1% agarose gel, by staining with ethidium bromide and observed under UV light.

Cloning

Positive PCR amplification products sequenced in bidirectional displayed double peaks. To confirm that the sequencing reaction from the PCR product was not affected by the presence of contaminating PCR products, PCR products were purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, China), according to the manufacturer’s instructions. Positive products were cloned into a 5minTM TA/Blunt-Zero Cloning Kit (Vazyme Biotech Co., Ltd.) and used to transform Escherichia coli DH5α cells. Clones were selected on LuriaBertani (LB) agar supplemented with 50 μg/mL of ampicillin.

Sequence and phylogenetic analysis

All positive clones were sequenced from the vector and identified the species of trichomonads. These recombinant clones were sequenced unidirectionally at Tsingke Biotech Co., Ltd., Beijing, China. The primers used for sequencing were M13F (5′-GTAAAACGACGGCCAGT-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′). Nucleotide sequences were assembled with ChromasPro2.1.6 (https://www.technelysium.com.au/ChromasPro.html), edited using BioEdit 7.1.3 (https://www.mbio.ncsu.edu/BioEdit/bioedit.html). Nucleotide sequences were aligned with the reference sequences from GenBank using ClustalX 2.1 software (https://www.clustal.org). Phylogenetic relationships among the species were evaluated using neighbor-joining analyses in MEGA 11 (https://www.megasoftware.net/), with branch reliability assessed by 1,000 bootstrap replicates. A phylogenetic tree representing the flagellated protozoa was then constructed. Nucleotide sequences generated in the study were submitted to GenBank under accession numbers PP829134–PP829172 and PP829309–PP829318. Three sequences failed to upload to NCBI and are presented as Supplementary File 1.

Statistical analysis

The chi-square (χ2) test, conducted using IBM SPSS Statistics 22 software (International Business Machines Corp., New York, NY, USA), was used to compare differences in trichomonad colonization rates across different living conditions of Milu deer. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to identify the risk factors associated with the occurrence of these pathogens in Milu deer. Differences were considered statistically significant at p < 0.05.

Results

Occurrence of and risk factors for trichomonads in random sampling

Among the 112 fecal samples collected, PCR testing made it possible to identify 43 as positive for trichomonads (Table 2). The occurrence ranged from 19.23% to 100% in different provinces. Data analysis revealed that Milu deer in Hubei (100%, 12/12) had a significantly higher detection rate compared to those in Beijing (26.08%, 12/46; χ2 = 21.435, p < 0.001), Inner Mongolia (19.23%, 5/26; χ2 = 21.665, p < 0.001), Hebei (46.15%, 6/13; χ2 = 8.974, p = 0.003), and Jiangsu (53.33%, 8/15; χ2 = 7.560, p = 0.006). Significant differences in colonization rates observed between Jiangsu (53.33%, 8/15) and Inner Mongolia (19.23%, 5/26; χ2 = 5.109, p = 0.024); however, differences with Beijing (26.08%, 12/46; χ2 = 3.811, p = 0.051), and Hebei (46.15%, 6/13; χ2 = 0.144, p = 0.705) were not statistically significant. In addition, there were no significant impacts on the colonization rates in Beijing (26.08%, 12/46), Hebei (46.15%, 6/13; χ2 = 1.925, p = 0.165), and Inner Mongolia (19.23%, 5/26; χ2 = 0.433, p = 0.511).

Furthermore, OR analysis revealed that Milu deer in Hubei exhibited a significantly higher risk of trichomonad colonization, with an occurrence of 100% (OR = 55.159, 95% CI = 3.166–961.113, p = 0.006), indicating notable vulnerability due to varying localities (Table 2). Results of the logistic regression analysis indicated that trichomonad colonization was not significantly associated with geographic coordinates, annual mean temperature, altitude, and number of individuals per hectare in the four Milu nature reserves (p > 0.05). However, there was an apparent association between trichomonad colonization with high relative humidity and average annual rainfall (OR = 11.675, 95% CI = 1.747–77.781, p = 0.011) (Figure 1).

thumbnail Figure 1

Association between trichomonad colonization and distribution of five Milu nature reserves in China, indicated by univariate analyses of data from this study. A potential risk factor for colonization was considered when p < 0.05 and OR > 1. *p < 0.05.

Distribution of trichomonad species

Among the 43 positive cloning products, four genera and 52 sequences from trichomonads were successfully identified via sequence analysis. Colonizations ranged from single to triple pathogen co-colonizations (Figure 2). Notably, 27 Milu deer were positive for single pathogen colonization, with occurrence rates for Simplicimonas spp. at 53.49%, and both Hypotrichomonas spp. and Hexamastix spp. at 4.65% each. Co-colonizations involving two pathogens occurred in 32.56% of cases (14/43), with combinations of Simplicimonas spp. and Hypotrichomonas spp. at 13.95%, Hexamastix spp. at 6.98%, and Tetratrichomonas spp. at 11.63%. Additionally, co-colonizations with three pathogens – Simplicimonas spp., Hypotrichomonas spp., and Hexamastix spp. – were observed in 2.33% of cases (1/43), similar to the co-colonization rate of Simplicimonas spp., Hypotrichomonas spp., and Tetratrichomonas spp. at 2.33% (1/43).

thumbnail Figure 2

Occurrence of trichomonad pathogens in Milu deer. Vertical columns show co-colonization rates in the same province. Single pathogen colonization, A1: Simplicimonas, A2: Hypotrichomonas, A3: Hexamastix. Co-colonizations with two pathogens, B1: Simplicimonas & Hypotrichomonas, B2: Simplicimonas & Hexamastix, B3: Simplicimonas & Tetratrichomonas. Co-colonizations with three pathogens, C1: Simplicimonas, Hypotrichomonas, & Hexamastix, C2: Simplicimonas, Hypotrichomonas, & Tetratrichomonas.

Across the sampling locations, only Simplicimonas spp. colonizations were found in the Milu deer in Jiangsu. However, five different combinations of four-pathogen colonizations occurred in Beijing and Hubei, respectively. Four types of colonization forms involving combinations of three pathogens, excluding Hypotrichomonas spp., were observed in Hebei. In Inner Mongolia, pathogens associated with Simplicimonas spp. and Hypotrichomonas spp. presented three different modes of colonization (Figure 1).

Genetic and phylogenetic analysis

No ITS-1/5.8S/ITS-2 sequences from trichomonads in Milu deer are available in GenBank; thus, phylogenetic analysis was conducted using comparable sequences (Figure 3). The analysis confirmed the identity of Hypotrichomonas spp., which shared 82.95–89.37% nucleotide identity with a strain isolated from a snake in Brazil (AY349192). The Tetratrichomonas spp. sequences were 89.47–96.01% identical to a strain isolated from a pig (AY886831). Additionally, the sequences of Simplicimonas spp., sharing 93.81–96.43% nucleotide identity, clustered in the clade with a strain isolated from cattle (KY410341). Furthermore, PP829141 showed a 99.67% nucleotide identity with a sequence from a chamaeleon (GQ254636). Moreover, three Hexamastix spp. sequences, not yet assigned NCBI accession numbers, exhibited 87.07–87.39% nucleotide similarity with a sequence from a rat (AB931162) and 91.97–92.36% with a sequence from a chamaeleon (AY319275). In order to rationalize Figure 3, we present a more general phylogenetic analysis tree covering a wider range of trichomonads as Supplementary File 2.

thumbnail Figure 3

Phylogenetic analysis of trichomonads based on the ITS1-5.8S rRNA-ITS2 gene. Sequences were retrieved from GenBank, aligned using ClustalW, and analyzed using MEGA 11 software. The neighbor-joining method was used to construct the trees from the Kimura-2-parameter model. Branch numbers represent percent bootstrapping values from 1,000 replicates, with values of more than 60% shown in the tree. The species identified in this study are indicated by ▲.

Discussion

Traditionally, microscopic examination of trichomonas in feces has been considered the gold standard [31–33]. However, differentiating between trichomonas species with similar morphological characteristics is often impractical in field conditions when examining Milu deer feces. Therefore, more sensitive PCR methods have been adopted for the detection and identification of trichomonas species. Although many trichomonads function as harmless intestinal symbionts, others, such as P. hominis, T. gallinae, T. gallinarum, and T. foetus, can cause significant diseases in livestock and are critically important in veterinary medicine [16, 26, 28, 38, 39]. Identifying the specific trichomonad species in Milu deer is therefore crucial and contributes valuable insights for the ex-situ conservation of this species.

In the current work, we investigated the occurrence of trichomonad in Milu deer via amplification of the ITS1-5.8S rRNA-ITS2 gene. The overall colonization rate in Milu deer was 38.39% (43/112). Globally, trichomonas infections are prevalent in various hosts. In pigs, the infection rates were reported at 52.08% (100/192) in Austria [40], 43.04% (68/158) in Changchun, and 47.4% (237/500) in Anhui, China [24, 31]. For non-human primates (NHPs), the rates were 46.7% (28/60) in North China [34], 27.6% (45/163) among gorillas in the Central African Republic [41], 68.6% (48/70) in wild chimpanzees in Uganda [43], and 31.3% (5/16) in southeast Brazil [15]. These findings underscore the extensive and significant occurrence of trichomonads across diverse host populations. Thus, detecting trichomonad colonization in Milu deer is significant, as it further expands the host range of trichomonads.

The occurrence of trichomonad colonization varied significantly across different nature reserves in this study. In Hubei, the occurrence (100%, 12/12) and risk of trichomonad colonization (OR = 55.159, 95% CI = 3.166–961.113, p = 0.006) were markedly higher than in other regions (Table 2). This variation in colonization rates may be attributable to the environmental conditions [36, 52]. Factors such as the higher relative humidity and average annual rainfall in Hubei likely contribute to elevated colonization rates. A logistic regression analysis of the distribution summaries of the five nature reserves confirms that the high humidity and average annual rainfall (OR = 11.675, 95% CI = 1.747–77.781, p = 0.011) in Hubei probably contributed to the high colonization rate of Milu deer (Figure 1). Additionally, the size and density of Milu deer herds in nature reserves could facilitate the accumulation of parasites in the environment [46]. Stagnant water in the marsh contaminates the environment. Milu deer become carriers of parasites due to the contaminated environment, spreading the colonization through fecal-oral transmission and increasing the risk of trichomonad colonization. Finally, the limited number of samples collected might also have influenced the observed differences in trichomonad occurrence across various regions.

Four species of trichomonads were found in Milu deer (Figure 2). Although some trichomonads may have been traditionally viewed as parasites, recent studies suggest that they may also act as mutualists, contributing to the host’s health and gut homeostasis. For instance, the genus Tritrichomonas can help regulate the gut microbiome by promoting microbial diversity or enhancing nutrient absorption, benefiting the host in a symbiotic manner [42]. This mutualistic relationship could lead to improved digestive efficiency, better immune function, and protection against other pathogens by maintaining a stable gut microbiota.

Simplicimonas spp., members of the class Tritrichomonadea, are typically considered commensals of the intestinal tract due to their non-pathogenic nature [14, 20]. However, this genus has been found in a variety of hosts including livestock, birds, rodents, and reptiles, sometimes in association with T. gallinarum. Although Simplicimonas spp. are generally regarded as low pathogenicity species that exhibit host specificity among avians, they occasionally present as granulomatous disease [29]. Given the widespread distribution of Simplicimonas spp. in the Milu deer, it is plausible that these organisms may have a similar role as commensals or mutualists in this host species. Further research should aim to clarify the functional roles of Simplicimonas spp. in the gut ecosystem of the Milu deer and examine whether their presence contributes to positive outcomes, such as improved gut health or metabolic processes.

Tetratrichomonas spp. comprises approximately ten valid species and is a common parasite that readily transmits, infecting various vertebrates, including humans, ruminants, birds, rodents, and NHPs under favorable conditions [3, 18, 37]. This genus has been identified as an opportunistic pathogen. Previous studies have indicated that an overgrowth of Tetratrichomonas spp. in cattle can lead to diarrhea, causing significant economic losses in domestic animals and adversely affecting the health of wild animals [3, 17]. Consequently, it is essential to monitor Milu deer for Tetratrichomonas spp. regularly to prevent and control potential diseases.

Hypotrichomonas spp., a relatively obscure genus within the class Hypotrichomonadida, has been reported in NHPs, reptiles, and rodents [12]. Similarly, species of Hexamastix, belonging to the class Honigbergiellida, have been known to infect reptiles and rodents [25, 44]. In this study, we detected both Hypotrichomonas spp. and Hexamastix spp. in Milu deer, thereby broadening their known host range. However, the scant information on the pathogenic roles of Hypotrichomonas spp. and Hexamastix spp. complicates the determination of their effects in these hosts. Further research is required to understand their life cycles and pathogenic potential.

Hypotrichomonas spp. and Hexamastix spp., detected in Milu deer, clustered in a separate clade rather than with the reference sequences, while Simplicimonas spp. and Tetratrichomonas spp. predominantly aligned with the reference sequences (Figure 3). This divergence may stem from the limited host range of Hypotrichomonas spp. and Hexamastix spp., as current studies have identified only a few strains. Conversely, Simplicimonas spp. and Tetratrichomonas spp. are known to infect a wide variety of hosts [3, 14, 18, 20]. Notably, none of the sequences were 100% homologous with those in the NCBI during comparisons, suggesting distinct genetic profiles of trichomonads in Milu deer compared to those in other hosts. Interestingly, phylogenetic analysis revealed that Tetratrichomonas spp. and Hypotrichomonas spp. from the same nature reserves clustered in a single clade. This may indicate variability in Milu deer intestinal trichomonads across nature reserves. Consequently, more comprehensive evolutionary analyses and studies on trichomonads in Milu deer are imperative to enhance the accuracy of parasitism detection and inform the development of effective prevention and treatment strategies.

Furthermore, previous studies demonstrated the existence of genetically distinct mixed infections of T. gallinae in individual animals [22]. In our study, based on the species differences of trichomonads, mixed colonizations were considered to be present in Milu deer. Consequently, management of living conditions and regular monitoring of trichomonad numbers, along with the implementation of treatments for affected individuals, is critical to ensure the health of Milu deer. The small sample size and the limited number of sample collection sites in this study might suggest the presence of other trichomonad species in Milu deer that have not yet been recognized. Additional regions and samples are necessary to elucidate the potential pathogenicity of trichomonads in various mucosae to assess their clinical and public health impacts.

Conclusion

The results of this study reveal a high occurrence of trichomonads in Milu deer, identifying sample collection sites, high relative humidity, and high mean annual rainfall as significant risk factors for colonization. Trichomonads might threaten the health of Milu deer and are probably transmitted from other species. To mitigate the occurrence of trichomonad colonization, it is crucial to implement effective management strategies that minimize environmental contamination and enhance the health and welfare of Milu deer.

Funding

This work was supported by the Financial Program of BJAST (25CA005/24CA003), the Natural Science Research Project for Anhui Universities (2024AH050434), and the Natural Science Foundation of Anhui Province (2008085QC136).

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author contribution statement

Y.L.Z. and Z.C.L conceived and wrote the project, collected and analyzed the data, and wrote the manuscript. X.L.S. and L.R.H. carried out data collection and verification and revised the manuscript. G.D.X. and L.J.T. participated in data collection and verification and revised the manuscript. C.Y. and Z.L. performed data curation. J.D.B and Z.Y.Z. participated in project management and revised the manuscript. Q.Y.G., C.S.Y. and Q.X.Z. managed the project and reviewed the manuscript. All authors approved the publication of this version of the manuscript.

Supplementary material

Supplementary File 1: Three sequences of Hexamastix sp. in Milu deer feces failed to upload to NCBI. Access here

Supplementary File 2: Phylogenetic analysis of trichomonads based on the ITS1-5.8S rRNA-ITS2 gene. Sequences were retrieved from GenBank, aligned using ClustalW, and analyzed using the MEGA 11 software. The neighbor-joining method was used to construct the trees from the Kimura-2-parameter model. Branch numbers represent percent bootstrapping values from 1,000 replicates, with values of more than 60% shown in the tree. The species identified in this study are indicated by ▲. In order to rationalize Figure 3, we present a more general phylogenetic analysis tree covering a wider range of trichomonad. Access here

References

  1. Adl SM, Bass D, Lane CE, Lukeš J, Schoch CL, Smirnov A, Agatha S, Berney C, Brown MW, Burki F, Cárdenas P, Čepička I, Chistyakova L, Del Campo J, Dunthorn M, Edvardsen B, Eglit Y, Guillou L, Hampl V, Heiss AA, Hoppenrath M, James TY, Karnkowska A, Karpov S, Kim E, Kolisko M, Kudryavtsev A, Lahr DJG, Lara E, Le Gall L, Lynn DH, Mann DG, Massana R, Mitchell EAD, Morrow C, Park JS, Pawlowski JW, Powell MJ, Richter DJ, Rueckert S, Shadwick L, Shimano S, Spiegel FW, Torruella G, Youssef N, Zlatogursky V, Zhang Q. 2019. Revisions to the classification, nomenclature, and diversity of eukaryotes. Journal of Eukaryotic Microbiology, 66, 4–119. [CrossRef] [PubMed] [Google Scholar]
  2. Amin A, Bilic I, Liebhart D, Hess M. 2014. Trichomonads in birds – a review. Parasitology, 141, 733–747. [CrossRef] [PubMed] [Google Scholar]
  3. Bailey NP, Velo-Rego E, Hirt RP. 2021. Sporadic isolation of Tetratrichomonas species from the cattle urogenital tract. Parasitology, 148, 1339–1344. [CrossRef] [PubMed] [Google Scholar]
  4. Bellanger AP, Cabaret O, Costa JM, Foulet F, Bretagne S, Botterel F. 2008. Two unusual occurrences of trichomoniasis: rapid species identification by PCR. Journal of Clinical Microbiology, 46, 3159–3161. [CrossRef] [PubMed] [Google Scholar]
  5. Bilic I, Jaskulska B, Souillard R, Liebhart D, Hess M. 2014. Multi-locus typing of Histomonas meleagridis isolates demonstrates the existence of two different genotypes. PLoS One, 9, e92438. [CrossRef] [PubMed] [Google Scholar]
  6. Carter JE, Whithaus KC. 2008. Neonatal respiratory tract involvement by Trichomonas vaginalis: a case report and review of the literature. American Journal of Tropical Medicine and Hygiene, 78, 17–19. [CrossRef] [PubMed] [Google Scholar]
  7. Cavalier-Smith T. 2013. Early evolution of eukaryote feeding modes, cell structural diversity, and classification of the protozoan phyla Loukozoa, Sulcozoa, and Choanozoa. European Journal of Protistology, 49, 115–178. [CrossRef] [PubMed] [Google Scholar]
  8. Cepicka I, Dolan MF, Gile G. 2017. Parabasalia, in Handbook of the Protists, Archibald J, Simpson A, Slamovits C, Editors. Springer International Publishing, Cham, p. 1175–1218. [CrossRef] [Google Scholar]
  9. Cepicka I, Hampl V, Kulda J.2010. Critical taxonomic revision of Parabasalids with description of one new genus and three new species. Protist, 161, 400–433. [CrossRef] [PubMed] [Google Scholar]
  10. Cepicka I, Hampl V, Kulda J, Flegr J. 2006. New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas. Molecular Phylogenetics and Evolution, 39, 542–551. [CrossRef] [PubMed] [Google Scholar]
  11. Céza V, Kotyk M, Kubánková A, Yubuki N, Šťáhlavský F, Silberman JD, Čepička I. 2022. Free-living Trichomonads are unexpectedly diverse. Protist, 173, 125883. [CrossRef] [PubMed] [Google Scholar]
  12. Céza V, Pánek T, Smejkalová P, Čepička I. 2015. Molecular and morphological diversity of the genus Hypotrichomonas (Parabasalia: Hypotrichomonadida), with descriptions of six new species. European Journal of Protistology, 51, 158–172. [CrossRef] [PubMed] [Google Scholar]
  13. Chen YQ, Zhang QX, Cheng ZB, Shan YF, Pu TC, Liu T, Guo QY, Zhang P, Song XL, Yang CS, Zhong ZY, Bai JD. 2024. First screening for tick-borne pathogens in Chinese Milu deer (Elaphurus davidianus), Ticks and Tick-Borne Diseases, 15, 102293. [CrossRef] [PubMed] [Google Scholar]
  14. Dimasuay KGB, Lavilla OJY, Rivera WL. 2013. New hosts of Simplicimonas similis and Trichomitus batrachorum identified by 18S ribosomal RNA gene sequences. Journal of Parasitology Research, 2013, 831947. [PubMed] [Google Scholar]
  15. Dos Santos CS, de Jesus VLT, McIntosh D, Carreiro CC, Batista LCO, do Bomfim Lopes B, Neves DM, Lopes CWG. 2017. Morphological, ultrastructural, and molecular characterization of intestinal tetratrichomonads isolated from non-human primates in southeastern Brazil. Parasitology Research, 116, 2479–2488. [CrossRef] [PubMed] [Google Scholar]
  16. Duboucher C, Mogenet M, Périé G. 1995. Salivary trichomoniasis. A case report of infestation of a submaxillary gland by Trichomonas tenax. Archives of Pathology & Laboratory Medicine, 119, 277–279. [PubMed] [Google Scholar]
  17. Dufernez F, Walker RL, Noël C, Caby S, Mantini C, Delgado-Viscogliosi P, Ohkuma M, Kudo T, Capron M, Pierce RJ, Villanueva MR, Viscogliosi E. 2007. Morphological and molecular identification of non-Tritrichomonas foetus trichomonad protozoa from the bovine preputial cavity. Journal of Eukaryotic Microbiology, 54, 161–168. [CrossRef] [PubMed] [Google Scholar]
  18. Falkowski P, Liebhart D, Bobrek K, Gaweł A. 2020. The prevalence of Tetratrichomonas spp. in reproductive geese flocks. Avian Diseases, 64, 547–551. [CrossRef] [PubMed] [Google Scholar]
  19. Frey CF, Müller N. 2012. Tritrichomonas – systematics of an enigmatic genus. Molecular and Cellular Probes, 26, 132–136. [CrossRef] [PubMed] [Google Scholar]
  20. Frey CF, Müller N, Stäuber N, Marreros N, Hofmann L, Hentrich B, Hirsbrunner G. 2017. Simplicimonas-like DNA in vaginal swabs of cows and heifers cross-reacting in the real-time PCR for T. foetus. Veterinary Parasitology, 237, 30–36. [CrossRef] [PubMed] [Google Scholar]
  21. Gilbert C, Ropiquet A, Hassanin A. 2006. Mitochondrial and nuclear phylogenies of Cervidae (Mammalia, Ruminantia): systematics, morphology, and biogeography. Molecular Phylogenetics and Evolution, 40, 101–117. [CrossRef] [PubMed] [Google Scholar]
  22. Grabensteiner E, Bilic I, Kolbe T, Hess M. 2010. Molecular analysis of clonal trichomonad isolates indicate the existence of heterogenic species present in different birds and within the same host. Veterinary Parasitology, 172, 53–64. [CrossRef] [PubMed] [Google Scholar]
  23. Huang SY, Fan YM, Yang Y, Ren YJ, Gong JZ, Yao N, Yang B. 2020. Prevalence and molecular characterization of Cryptosporidium spp. in Père David’s deer (Elaphurus davidianus) in Jiangsu, China. Revista Brasileira de Parasitologia Veterinaria, 29, e017919. [CrossRef] [PubMed] [Google Scholar]
  24. Huby-Chilton F, Scandrett BW, Chilton NB, Gajadhar AA. 2009. Detection and identification of Tetratrichomonas in a preputial wash from a bull by PCR and SSCP. Veterinary Parasitology, 166, 199–204. [CrossRef] [PubMed] [Google Scholar]
  25. Janakidevi K. 1961. A new species of Hexamastix (protozoa) parasitic in the spiny-tailed lizard, Uromastix hardwickii, Zeitschrift für Parasitenkunde, 21, 151–154. [Google Scholar]
  26. Johnson EH, Windsor JJ, Clark CG. 2004. Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clinical Microbiology Reviews, 17, 553–570. [CrossRef] [PubMed] [Google Scholar]
  27. Kim YA, Kim HY, Cho SH, Cheun HI, Yu JR, Lee SE. 2010. PCR detection and molecular characterization of Pentatrichomonas hominis from feces of dogs with diarrhea in the Republic of Korea. Korean Journal of Parasitology, 48, 9–13. [CrossRef] [PubMed] [Google Scholar]
  28. Kissinger P, Adamski A. 2013. Trichomoniasis and HIV interactions: a review. Sexually Transmitted Infections, 89, 426–433. [CrossRef] [PubMed] [Google Scholar]
  29. Landman WJM, Gantois N, Sawant M, Majoor FA, van Eck JHH, Viscogliosi E. 2021. Prevalence of trichomonads in the cloaca of wild wetland birds in the Netherlands. Avian Pathology, 50, 465–476. [CrossRef] [PubMed] [Google Scholar]
  30. Li J, Bai B, Meng Q, Cheng Z, Shan Y, Liu T, Zhong Z. 2023. Bacteria community diversity of feces of Père David’s Deer analyzed by Illumina MiSeq. Chinese Journal of Wildlife, 44, 98–105. (In Chinese). [Google Scholar]
  31. Li W, Li W, Gong P, Zhang C, Yang J, Zhang X, Li J. 2015. The prevalence of intestinal trichomonads in Chinese pigs. Veterinary Parasitology, 211, 12–15. [CrossRef] [PubMed] [Google Scholar]
  32. Li WC, Wang K, Li Y, Zhao LP, Xiao Y, Gu YF. 2018. Survey and molecular characterization of Trichomonads in pigs in Anhui province, east China, 2014. Iranian Journal of Parasitology, 13, 602–610. [PubMed] [Google Scholar]
  33. Li WC, Wang K, Zhang W, Wu J, Gu YF, Zhang XC. 2016. Prevalence and molecular characterization of intestinal Trichomonads in pet dogs in east China. Korean Journal of Parasitology, 54, 703–710. [Google Scholar]
  34. Li WC, Ying M, Gong PT, Li JH, Yang J, Li H, Zhang XC. 2016. Pentatrichomonas hominis: prevalence and molecular characterization in humans, dogs, and monkeys in northern China. Parasitology Research, 115, 569–574. [CrossRef] [PubMed] [Google Scholar]
  35. Lollis L, Gerhold R, McDougald L, Beckstead R. 2011. Molecular characterization of Histomonas meleagridis and other parabasalids in the United States using the 5.8S, ITS-1, and ITS-2 rRNA regions. Journal of Parasitology, 97, 610–615. [CrossRef] [PubMed] [Google Scholar]
  36. Ma PP, Zou Y, Mu WJ, Zhang YY, Li YQ, Liu ZL, Zhang L, Chen LX, Liu GH, Wang S. 2024. Prevalence of intestinal trichomonads in captive non-human primates in China. Parasite, 31, 19. [CrossRef] [EDP Sciences] [PubMed] [Google Scholar]
  37. Mantini C, Souppart L, Noël C, Duong TH, Mornet M, Carroger G, Dupont P, Masseret E, Goustille J, Capron M, Duboucher C, Dei-Cas E, Viscogliosi E. 2009. Molecular characterization of a new Tetratrichomonas species in a patient with empyema. Journal of Clinical Microbiology, 47, 2336–2339. [CrossRef] [PubMed] [Google Scholar]
  38. Maritz JM, Land KM, Carlton JM, Hirt RP. 2014 . What is the importance of zoonotic trichomonads for human health? Trends in Parasitology, 30, 333–341. [CrossRef] [PubMed] [Google Scholar]
  39. Meloni D, Mantini C, Goustille J, Desoubeaux G, Maakaroun-Vermesse Z, Chandenier J, Gantois N, Duboucher C, Fiori PL, Dei-Cas E, Duong TH, Viscogliosi E. 2011. Molecular identification of Pentatrichomonas hominis in two patients with gastrointestinal symptoms. Journal of Clinical Pathology, 64, 933–935. [CrossRef] [PubMed] [Google Scholar]
  40. Mostegl MM, Richter B, Nedorost N, Maderner A, Dinhopl N, Weissenböck H. 2011. Investigations on the prevalence and potential pathogenicity of intestinal trichomonads in pigs using in situ hybridization. Veterinary Parasitology, 178, 58–63. [CrossRef] [PubMed] [Google Scholar]
  41. Petrželková KJ, Smejkalová P, Céza V, Pafčo B, Shutt-Phillips KA, Todd A, Jirků-Pomajbíková K, Benavides J, Modrý D, Čepička I. 2020. Sympatric western lowland gorillas, central chimpanzees and humans are infected with different trichomonads. Parasitology, 147, 225–230. [CrossRef] [PubMed] [Google Scholar]
  42. Popovic A, Cao EY, Han J, Nursimulu N, Alves-Ferreira EVC, Burrows K, Kennard A, Alsmadi N, Grigg ME, Mortha A, Parkinson J. 2024. Commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota. ISME Journal, 18, wrae023. [CrossRef] [PubMed] [Google Scholar]
  43. Rushmore J, Allison AB, Edwards EE, Bagal U, Altizer S, Cranfield MR, Glenn TC, Liu H, Mudakikwa A, Mugisha L, Muller MN, Stumpf RM, Thompson ME, Wrangham R, Yabsley MJ. 2015. Screening wild and semi-free ranging great apes for putative sexually transmitted diseases: evidence of Trichomonadidae infections. American Journal of Primatology, 77, 1075–1085. [CrossRef] [PubMed] [Google Scholar]
  44. Tuska-Szalay B, Kelly H, Takács N, Kontschán J, Votýpka J, Hornok S. 2022. Molecular evidence of Monocercomonas and Acanthamoeba in the feces of captive reptiles. Parasitology Research, 121, 3681–3687. [CrossRef] [PubMed] [Google Scholar]
  45. Xie F, Zhang Z, Zhao A, Jing B, Qi M, Wang R. 2019. Molecular characterization of Cryptosporidium and Enterocytozoon bieneusi in Père David’s deer (Elaphurus davidianus) from Shishou, China. International Journal for Parasitology: Parasites and Wildlife, 10, 184–187. [CrossRef] [Google Scholar]
  46. Xu S, Zhang S, Hu X, Zhang B, Yang S, Hu X, Liu S, Hu D, Bai J. 2021. Temporal and spatial dynamics of gastrointestinal parasite infection in Père David’s deer. PeerJ, 9, e11335. [CrossRef] [PubMed] [Google Scholar]
  47. Yang D, Song Y, Ma J, Li P, Zhang H, Price MRS, Li C, Jiang Z. 2016. Stepping-stones and dispersal flow: establishment of a meta-population of Milu (Elaphurus davidianus) through natural re-wilding. Scientific Reports, 6, 27297. [CrossRef] [PubMed] [Google Scholar]
  48. Zhang H, Li P, Wen H, Tian G, Chen H, Zhang L, Zhu J. 2019. Population status and research progress of Père David’s Deer (Elaphurus davidianus) in China. Pakistan Journal of Zoology, 51, 1–9. [Google Scholar]
  49. Zhang M, Shi M, Fan M, Xu S, Li Y, Zhang T, Cha M, Liu Y, Guo X, Chen Q, Li Y, Zhang S, Hu D, Liu S. 2018. Comparative analysis of gut microbiota changes in Père David’s Deer populations in Beijing Milu Park and Shishou, Hubei province in China. Frontiers in Microbiology, 9, 1258. [CrossRef] [PubMed] [Google Scholar]
  50. Zhang Q, Zhong Z, Xia Z, Meng Q, Shan Y, Guo Q, Cheng Z, Zhang P, He H, Bai J. 2022. Molecular epidemiology and genetic diversity of Enterocytozoon bieneusi in cervids from Milu Park in Beijing, China. Animals, 12, 1539. [CrossRef] [PubMed] [Google Scholar]
  51. Zhang Y, Bai J, Zhu A, Chen R, Xue D, Zhong Z, Cheng Z. 2021. Reversing extinction in China’s Père David’s deer. Science, 371, 685. [CrossRef] [Google Scholar]
  52. Zhang Y, Lu Z, Liu Z, Zhou Y, Xiao G, Opeyemi AO, Jin S, Li Y, Liu T, Wu Q, Sun X, Xu Q, Zhang Q, Yang C. 2024. Prevalence and molecular characterization of Trichomonas gallinae from pigeons in Anhui, China. Comparative Immunology, Microbiology and Infectious Diseases, 107, 102157. [CrossRef] [PubMed] [Google Scholar]

Cite this article as: Zhang Y, Li Z, Song X, Xiao G, He L, Bai J, Zhong Z, Tian L, Chang Y, Li Z, Guo Q, Yang C & Zhang Q. 2025. Genetic diversity of Trichomonads from Milu deer (Elaphurus davidianus) in China. Parasite 32, 23. https://doi.org/10.1051/parasite/2025015.

All Tables

Table 1

Summary of characteristics of the five Milu nature reserves in China.

In the text
Table 2

Occurrence of trichomonad colonization in Milu deer. A potential risk factor for colonization was considered when p < 0.05 and OR > 1.

In the text

All Figures

thumbnail Figure 1

Association between trichomonad colonization and distribution of five Milu nature reserves in China, indicated by univariate analyses of data from this study. A potential risk factor for colonization was considered when p < 0.05 and OR > 1. *p < 0.05.
In the text
thumbnail Figure 2

Occurrence of trichomonad pathogens in Milu deer. Vertical columns show co-colonization rates in the same province. Single pathogen colonization, A1: Simplicimonas, A2: Hypotrichomonas, A3: Hexamastix. Co-colonizations with two pathogens, B1: Simplicimonas & Hypotrichomonas, B2: Simplicimonas & Hexamastix, B3: Simplicimonas & Tetratrichomonas. Co-colonizations with three pathogens, C1: Simplicimonas, Hypotrichomonas, & Hexamastix, C2: Simplicimonas, Hypotrichomonas, & Tetratrichomonas.
In the text
thumbnail Figure 3

Phylogenetic analysis of trichomonads based on the ITS1-5.8S rRNA-ITS2 gene. Sequences were retrieved from GenBank, aligned using ClustalW, and analyzed using MEGA 11 software. The neighbor-joining method was used to construct the trees from the Kimura-2-parameter model. Branch numbers represent percent bootstrapping values from 1,000 replicates, with values of more than 60% shown in the tree. The species identified in this study are indicated by ▲.
In the text

Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.

Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.

Initial download of the metrics may take a while.