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All issues Volume 32 (2025) Parasite, 32 (2025) 22 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 22
Number of page(s) 10
DOI https://doi.org/10.1051/parasite/2025016
Published online 09 April 2025

© X.-C. Jiang 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

Trichomoniasis is a widespread parasitic disease affecting various animals and humans. In dogs and cats, Tritrichomonas foetus and Pentatrichomonas hominis are two trichomonad species, both classified within the Trichomonadidae family [13, 21, 36]. Tritrichomonas foetus is a dangerous pathogen to many kinds of animal host, including cattle, dogs, cats and pigs. Literature reports have indicated that T. foetus might have the capability to infect humans, posing significant potential for zoonotic disease [28]. It has been reported that T. foetus is an important cause of chronic and stubborn diarrhea in dogs and cats, parasitizing in the intestines [11, 18]. Pentatrichomonas hominis is commonly recognized as a human parasite that parasitizes the intestines of immunocompromized patients and mammals, causing diarrhea [39]. This species exhibits a broad host range including humans, dogs, goats, pigs, monkeys, cattle, cats and farmed wildlife [25, 30]. Immunocompromized patients infected with P. hominis typically exhibit symptoms including diarrhea, fever, nausea and vomiting, abdominal pain, bloating and appetite loss [6, 30]. Both T. foetus and P. hominis exhibit a single form, the trophozoite, throughout the entire life cycle. Both can be transmitted via the fecal-oral route, while T. foetus can also be sexually transmitted. In addition, T. foetus can parasitize the tissues of the reproductive tract, leading to infertility and abortion occasionally [13, 35].

Nowadays, companion animals are assuming an ever-growing significance in human lives. However, trichomoniasis in dogs and cats can lead to economic losses and pose potential zoonotic risks to humans. Currently, there remains a limited number of studies on trichomoniasis in dogs and cats, especially P. hominis. Traditionally, the detection of trichomonad infection relies on the fecal floating method, such as microscopic examinations, isolation and cultivation of insect strains, and electron microscopy. In fact, these methods were unable to accurately differentiate between P. hominis with T. foetus. Polymerase chain reaction (PCR) has been widely used to identify Trichomonas species by amplifying specific genes [29, 34]. Thus, the present study aimed to determine the prevalence and evaluate the risk factors associated with trichomonad infection in dogs and cats in Nanchang city, Jiangxi province, China. The findings provide new insights into the epidemiology of trichomonads in dogs and cats in south China.

Materials and methods

Ethics statement

This experiment was conducted in strict accordance with the experimental animal regulations of Jiangxi Agricultural University. All specimens were collected by anal swab with the consent of the pet owner, and the whole sampling process did not cause damage to animals.

Specimen collection

A total of 405 stool samples, including 111 from cats and 286 from dogs were collected from a pet hospital, a police dog base, and the training base of Jiangxi Agricultural University and a stray dog shelter in the Economic Development Zone of Nanchang City between 2020 and 2023 (Fig. 1). Each fecal sample was collected into a 2 mL stool sampling pipe and labeled with breed, age, gender, collecting location and sampling time. All samples were suitably placed in a sampling box filled with ice packs, and finally stored at −80 °C until DNA extraction.

thumbnail Figure 1

Location of sampling sites.

Microscopic examination

Each fecal sample was subjected to stool smear preparation, followed by staining using the Giemsa method. The stained smears were then examined under an optical microscope to detect the presence of Trichomonas and to observe its structural details.

DNA extraction

Each sample was cleaned with distilled water by centrifuging at 13,000 rpm for 5 min to remove redundant impurities before DNA extraction. Then, stool samples were extracted using an E.Z.N.A.® Stool DNA Kit (Omega Bio-Tek Inc, Norcross, GA, USA) and the extracted fecal DNA samples were divided and stored at −20 °C until PCR amplification.

PCR amplification

According to the research conducted by Felleisen et al. [10], Gookin et al. [13], and Li et al. [21], nested PCR primers were synthesized by Tsingke Biotechnology Co., Ltd. For P. hominis, the primers OF (ATG GCG AGT GGT GGA ATA) and OR (CCC AAC TAC GCT AAG GAT T) were used for the first round of PCR amplification, while the primers IF (TGT AAA CGA TGC CGA CAG AG) and IR (CAA CAC TGA AGC CAA TGC GAG C) were used for the second round. These primers were designed based on the 18S rRNA gene. Additionally, based on the sequence of ITS1-5.8SrRNA-ITS2 of T. foetus, the primers TF-F (CGT ATC AAG CAG GAG GAA GAG GG), TF-R (ATG CTT CAG TTC AGC GGG TCT TC), TF-R4 (CCT GCC GTT GGA TCA GTT TCG TTA A) and TF-R3 (CGG GTC TTC CTA TAT GAG ACA GAA CC) were designed for the nested PCR of T. foetus. The first round of the PCR reaction included genomic DNA (2 μL), 10× PCR buffer (Mg2+ plus) (2.5 μL), dNTP mixture (0.2 mM), primers (each primer 0.4 μM), Taq DNA polymerase (1.25 U), made up to 25 μL with double distilled water. The second round of PCR system was 25 μL including 17.3 μL sterilized double distilled water, 2.5 μL 10× PCR buffer (Mg2+ plus), 2 μL dNTP mixture (2.5 mmol/L), 1 μL upstream primer, 1 μL downstream primer, 0.2 μL Tag DNA polymerase (5 U/L), and 1 μL DNA template (first round amplification product). Annealing temperatures for the nested PCR of P. hominis were 59 °C and 61 °C, respectively. Annealing temperatures for T. foetus were 57 °C and 54 °C. Each reaction included a positive rate (DNA from T. foetus and P. hominis) and negative control (double distilled water). The second PCR products were examined by 1.5% (w/v) agarose gel electrophoresis and stained with Gelbule. PCR products of the right size were sequenced.

Sequence and phylogenetic analyses

Each of the positive PCR products were sent for bi-directional sequencing by Tsingke Biotechnology Co., Ltd. All obtained sequences were subjected to BLAST analysis in NCBI. This allowed for the determination of whether the specimens in this experiment were infected with P. hominis or T. foetus. Phylogenetic analysis was conducted based on the 18SrRNA gene of P. hominis and part of the ITS1-5.8SrRNA-ITS2 gene of T. foetus. Using the evolutionary tree drawing software MEGA11 and by the neighbor-joining method (NJ), with the bootstrap parameter set to 1,000, the branches having bootstrap values blow 50 were not viewed in evolutionary tree.

Statistical analysis

SPSS version 25.0 (IBM SPSS Inc., Chicago, IL, USA) was used to analyze the relationships between Trichomonas (T. foetus and P. hominis) prevalence and independent factors (breeds, gender, age and season) by the chi-square (χ2) test. It was considered that a difference in prevalence was significantly related to a factor when the p-value was less than 0.05. The infectious risk of T. foetus and P. hominis was also assessed in dogs and cats, considering various factors. The accuracy of the results was evaluated using odds ratios (ORs) and 95% confidence intervals (CIs).

Nucleotide sequence accession numbers

The representative nucleotide sequences were submitted to the GenBank database under accession numbers: PP930991–PP930994, PP932478–PP932483 and PP937742–PP937752.

Results

Microscopic Examination of T. foetus and P. hominis

In this study, a total of 294 fecal samples from dogs and 111 fecal samples from cats were stained and observed under a clinical light microscope, using the Giemsa staining method. The results showed that the positive rate of trichomoniasis was 10.20% (30/294) in dogs and 18.9% (21/111) in cats. The parasite was observed and mainly melon-shaped or oval and its size was similar to Trichomonas. There were five flagella in the front and back parts of the parasite, which were connected to the fluctuating membrane on the side of Trichomonas, and the length of flagella was approximately the same as the body (Figs. 2A and 2B). However, the morphologies under light microscopy did not allow us to differentiate the two species, T. foetus and P. hominis, effectively.

thumbnail Figure 2

Trichomonas foetus detected by light microscopy, Gram staining. A, in a cat fecal sample; B, in a dog fecal sample.

Prevalence of T. foetus and P. hominis

After sequencing identification, 17 dog fecal samples and 45 cat fecal samples tested for T. foetus were positive, with an overall prevalence of 15.3% (62/405), resulting in a prevalence of 5.78% (17/294) in dogs and 40.5% (45/111) in cats. For P. hominis, 66 dog samples and 4 cat samples were positive, with an overall prevalence of 17.3% (70/405), with a prevalence of 22.5% (66/294) in dogs and 3.6% (4/111) in cats. Statistical analysis showed that the prevalence of T. foetus in dogs was significantly correlated with breeds (p < 0.01), seasons (p < 0.01) and the environment (p < 0.01). In cats, the prevalence was significantly correlated with both season and age (p < 0.05) (Table 2). For P. hominis, the prevalence in dogs was significantly correlated with breeds and environments (p < 0.05) (Table 3). It is important to note that due to missing data for some samples, including most of the information on stray dogs and some details regarding the diarrhea status of both dogs and cats, these samples were not included in the calculations.

Table 1

Sample information.

Table 2

Distribution of T. foetus and P. hominis of dogs.

Table 3

Distribution of T. foetus and P. hominis in cats.

Phylogenetic Analysis of T. foetus and P. hominis

Phylogenetic analysis shows that the sequences of T. foetus in this research clustered into a large branch with isolates of T. foetus obtained from other animals with higher bootstrap values (Fig. 3). In the large branch, it is worth noting that three feline T. foetus isolates clustered on a small branch, while the other three feline T. foetus isolates and five canine T. foetus isolates clustered on a branch, indicating differences and mutual transmission of T. foetus between dogs and cats. In this study, P. hominis sequence all clustered into a large branch obtained from other animals, indicating that P. hominis for different animals had a closer relationship (Fig. 4). Meanwhile, Feline P. hominis and Canine P. hominis are mixed together, indicating the mutual transmission of P. hominis in dogs and cats, and even among other animals.

thumbnail Figure 3

Phylogenetic analysis of Trichomonas foetus using the neighbor-joining method (NJ) based on the ITS1-5.8SrRNA-ITS2 gene. The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with values >50% shown in the tree. Solid black triangles: species/subtypes identified in this study.

thumbnail Figure 4

Phylogenetic analysis of Pentatrichomonas hominis using the neighbor-joining method (NJ) based on the 18SrRNA gene. The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with values >50% shown in the tree. Solid black triangles: species/subtypes identified in this study.

Discussion

This study was the first examination of the prevalence of T. foetus and P. hominis in dogs and cats in Nanchang city, China. We found that the detection rate of microscopic examination was low, making it impossible to differentiate between T. foetus and P. hominis. Based on PCR, we found that the overall prevalence of T. foetus in dogs and cats in Jiangxi Province was 5.8% (17/294) and 40.5% (45/111), respectively. The results were in alignment with the current prevalent trend of T. foetus in dogs and cats in China, for which the prevalence in dogs ranged from 0.64% to 54.7%, and in cats ranged from 10.1% to 47.4% [17, 22, 24]. In other research, the prevalence of T. foetus in dogs was only 5.3% (2/38) in the United States [36], the prevalence of T. foetus in cats was 6.7% in South Korea [33], 1.39% (3/215) in Thailand [20], and 20.52% (24/117) in Poland [5], which is consistent with our study. Here, we found that the prevalence of canine T. foetus infection was higher in non-breed dogs (16.3%, 8/46) than purebred dogs (4.2%, 7/165). Nevertheless, no pertinent research exists to corroborate our findings. There is still a lack of evidence regarding whether breeds are predisposed to parasite infection at the genetic or immunological level. Our findings also indicated that the prevalence of canine T. foetus was related to season, for which the positive rate of canine T. foetus infection was higher in summer than in autumn and winter. The warm conditions of spring and summer enable parasites to thrive for an extended duration within moist feces [14], which may be conducive to spreading. Interestingly, we discovered that the living condition was also an important factor that influenced the prevalence of T. foetus, for which the positive rate in dogs in pet hospitals (11.5%, 15/131) was higher than at the police dog base (2.5%, 2/80). In this study, the findings regarding potential risk factors for canine T. foetus infection indicated that age, sex, and diarrhea status exhibited no significant differences, aligning closely with the observations reported in previous studies [12, 24].

Our results indicated a significant correlation between the young age of cats (<1 year old) and T. foetus infection, which was consistent with reports in Spain [31], the United Kingdom [16], Greece [37] and China [38]. Our results confirmed that the positive rate of feline T. foetus infection was higher in purebred cats (47.3%, 35/74) than non-purebred cats (27.0%, 10/37), for which the result was reversed for dogs and similar to the findings of other authors [36]. The elevated incidence of tritrichomonosis in purebred cats, predominantly residing in multi-cat households, may be caused by high density. This relationship heightens the risk of introducing parasites into cat houses and facilitates its further transmission [2]. Similarly, our investigation revealed that the prevalence of feline T. foetus infection was correlated with season. It was notable that the infection rates in spring, autumn and winter showed no significant differences, whereas in summer, the rate was the lowest. However, some reports found that the infection rate of feline T. foetus was higher in spring and summer than in winter. This might result from the uneven seasonal distribution of the samples in this study. Therefore, the association between the susceptibility of T. foetus and seasons is worthy of further research. Some studies have found that the susceptibility of feline T. foetus was not gender-biased [1, 3], and our results confirmed this. Diarrhea is a major clinical manifestation of feline T. foetus [1, 17]. Some studies have reported a significant correlation between diarrhea symptoms and feline T. foetus infection [7, 15], and there were also reports that indicated no significant difference between feline T. foetus infection and fecal status [5, 19]. In this study, there was also no significant correlation between diarrhea symptoms and feline T. foetus infection, which might be influenced by the sample size.

In this study, the prevalence of P. hominis in dogs and cats in Jiangxi province was 22.4% (66/294) and 3.6% (4/111), which was similar to the epidemic trend of canine P. hominis in the northern (27.4%, 69/252) and eastern (31.4%, 99/315) regions of China, and feline P. hominis infection was also consistent with previous research in China (5.3% 3/57) [5, 23]. The positive rate of P. hominis in dogs was 47.4% (18/38) in the United States, 15.8% (34/215) in France and 6.9% (38/544) in Japan; the prevalence of P. hominis in cats was 1.9% (2/103) in the United States, 17.7% (21/119) in Thailand and 0.5% (2/409) in Japan [26, 32], which is consistent with the findings of our study. Our results showed that the feeding environment had a certain effect on canine P. hominis infection, for which the prevalence rates in dogs in police service (31.3%, 26/83) and in street dogs (23.8%, 19/80) were higher than in pet hospital dogs (16.0%, 21/131). The stray dog rescue center and the police dog training facility could serve as gathering points for dogs, potentially facilitating the transmission of canine P. hominis. Conversely, the positive rate of canine P. hominis infection was relatively low among individual animals receiving treatment at pet hospitals. Our results also indicated that sex, age, breed, diarrhea manifestations and season did not significantly influence the infection rate of canine P. hominis in dogs. Meanwhile, no significant differences were found for factors of infection with feline P. hominis. Therefore, it remains essential to expand the sample size for further investigation.

Both T. foetus and P. hominis have an extensive host range [3, 9, 22]. First, T. foetus has been isolated from a variety of pets and farm animals, with the same strain known to infect cattle and pigs [27], but different genotypes infect cattle and cats [4, 8]; the origins of dog infections remain unclear. It was reported that more than two T. foetus genotypes capable of colonizing had an extensive range of hosts, including humans [9]. Second, P. hominis has been isolated from a variety of pets and farm animals [27], but little is known about its infection routes and epidemiology; the same strain could be circulating between all identified hosts. In this study, the phylogenetic analyses demonstrated that the sequences of P. hominis and T. foetus from the current study consistently cluster with their respective trichomonad counterparts. This clustering pattern suggested the potential for interspecies transmission between cats and dogs. Notably, a subset of feline T. foetus sequences had been observed to diverge from the main cluster, clustering closely with Tritrichomonas musculus, which is likely to be derived from another species. This divergence indicates that T. foetus might involve a risk of cross-species transmission.

Conclusions

This study investigated the prevalence of T. foetus and P. hominis, two causative agents of trichomoniasis, in dogs and cats in Nanchang city, south China. The overall prevalence of T. foetus was 15.3% (62/405), with a significantly higher prevalence in cats (40.5%, 45/111) compared to dogs (5.8%, 17/294). For P. hominis, the overall prevalence was 17.3% (70/405), with dogs having a higher prevalence (22.4%, 66/294) than cats (3.6%, 4/111). Statistical analysis revealed significant correlations between the prevalence of T. foetus and factors such as breed, season and environment (p < 0.01) in dogs, and with season and age (p < 0.05) in cats. Similarly, statistical analysis revealed significant correlations between the prevalence of P. hominis and environment (p < 0.05) in dogs. No factors were identified as being related to the prevalence of P. hominis in cats. The risk of trichomoniasis transmission in dogs and cats is significantly elevated in environments characterized by a high concentration of companion animals, such as breeding facilities and households with multiple companion animals. The phylogenetic analyses indicated that T. foetus might involve a risk of cross-species transmission. One of the limitations of this study was the sample size, which might not be sufficient to represent the entire population of dogs and cats in Nanchang City, China. Future studies should expand the sample size and cover more diverse geographical areas to provide a more comprehensive understanding of the pathogens. Moreover, incorporating long-term follow-up data and exploring the impact of various environmental factors on infection rates would enhance the understanding of the epidemiology of T. foetus and P. hominis in pets.

Funding

This work was supported by the College Student Innovation and Entrepreneurship Program Project (Grant No. S202410410002) and the Jiangxi Province Graduate Innovation Special Fund (Grant No. YC2024-S338).

Conflicts of interest

The authors declare that they have no competing interests.

Author contribution statement

XQC conceived and designed the study. XCJ and XT performed the experiments, analyzed the data and drafted the manuscript. YRM, STX, JJS and LLF participated in implementation of the study. XQC and YZ critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

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Cite this article as: Jiang X-C, Xiao T, Liu L-F, Ma Y-R, Xiao S-T, Shi J-J, Zou Y & Chen X-Q. 2025. Prevalence of Pentatrichomonas hominis and Tritrichomonas foetus in dogs and cats in Nanchang City, China. Parasite 32, 22. https://doi.org/10.1051/parasite/2025016.

All Tables

Table 1

Sample information.

In the text
Table 2

Distribution of T. foetus and P. hominis of dogs.

In the text
Table 3

Distribution of T. foetus and P. hominis in cats.

In the text

All Figures

thumbnail Figure 1

Location of sampling sites.
In the text
thumbnail Figure 2

Trichomonas foetus detected by light microscopy, Gram staining. A, in a cat fecal sample; B, in a dog fecal sample.
In the text
thumbnail Figure 3

Phylogenetic analysis of Trichomonas foetus using the neighbor-joining method (NJ) based on the ITS1-5.8SrRNA-ITS2 gene. The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with values >50% shown in the tree. Solid black triangles: species/subtypes identified in this study.
In the text
thumbnail Figure 4

Phylogenetic analysis of Pentatrichomonas hominis using the neighbor-joining method (NJ) based on the 18SrRNA gene. The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with values >50% shown in the tree. Solid black triangles: species/subtypes identified in this study.
In the text

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