兽研口腔为多种微生物提供了适宜的环境，包括毛滴虫等原虫，其可能与牙周病的发生相关。人被认为是口腔毛滴虫的典型宿主，但该虫也已在犬、猫及其他动物中发现，使其成为一种具有重要人兽共患潜力的病原。除口腔毛滴虫外，家养食肉动物还可能感染犬毛滴虫、猫毛滴虫和布氏毛滴虫等虫种。本研究旨在确定波兰犬和猫口腔毛滴虫定植的流行率、虫种组成及风险因素。采用培养法对238只家养动物（151只犬和87只猫）的口腔拭子进行了检查。对镜检阳性样本进行ITS1–5.8 S rRNA-ITS2片段的扩增，并对PCR产物进一步测序。151份犬样本中有26份（17.22%）、87份猫样本中有5份（5.75%）检出毛滴虫。在年龄较大的犬及牙结石严重的犬中观察到更高的检出频率。在犬和猫中检出的虫种仅为布氏毛滴虫和口腔毛滴虫。在相当数量的受检动物中检出人兽共患的口腔毛滴虫，凸显了这一发现对人类健康的重要意义。
引言
毛滴虫是广泛分布的原虫，存在于广泛宿主谱的不同部位。除其他部位外，它们已在人和多种动物（包括犬和猫）的口腔中检出（Dybicz et al. 2018; Nor Azmi et al. 2024; Matthew et al. 2025）。犬和猫的典型虫种为犬毛滴虫/猫毛滴虫或新近描述的布氏毛滴虫，但也发现了人兽共患虫种口腔毛滴虫（Kellerová and Tachezy 2017）。口腔毛滴虫的致病性广受讨论——它们常被认为无致病性，但大量研究证实其与牙周病相关（Patel et al. 2017; Kadhim et al. 2023; Matthew et al. 2023; Stoyanov et al. 2024）。已证实它们能够产生溶血素和蛋白酶，破坏胶原纤维并促进软组织降解（Nagao et al. 2000; Yamamoto et al. 2000）。它们也已在口腔之外检出，见于下颌下唾液腺、扁桃体和淋巴结。此外，它们还与肺部疾病及下呼吸道脓肿形成相关，尤其是在免疫功能低下或患有共存疾病的个体中（Szczepaniak et al. 2016; Kellerová and Tachezy 2017; Dybicz et al. 2018; Duboucher et al. 2000; Hong et al. 2024）。
尽管有明确证据表明其作为病原体的作用，毛滴虫在犬和猫中的认识仍很有限。这些家养动物因与人类密切接触而尤为重要，这增加了具有人兽共患潜力的病原传播风险。本研究旨在确定波兰犬和猫口腔中毛滴虫的流行率和虫种组成，以及动物年龄、口腔卫生状况和生活环境对病原流行率的影响。
材料与方法
材料
口腔拭子于2024年6月至2025年12月期间，在常规诊断和预防操作中，从来自波兰不同地区（下西里西亚、大波兰、罗兹省、波德拉谢省、波美拉尼亚、西波美拉尼亚省、卢布斯卡省、马佐夫舍、西里西亚、喀尔巴阡山）的151只家养犬和87只猫采集。研究纳入了不同品种的动物。使用无菌拭子从牙龈和牙齿表面单独采集样本，置于AMIES运输培养基（Deltalab，西班牙）中，并在采集后最长24小时内送至弗罗茨瓦夫环境与生命科学大学寄生虫学与侵袭性疾病教研室实验室。运输过程中，样本使用隔热包装以防止温度波动。动物按年龄（0–4岁、5–8岁和≥9岁）和牙结石严重程度分组（I级——无牙结石或牙结石覆盖牙面不超过25%，II级——牙结石覆盖牙面25%至50%，III级——牙结石覆盖牙面超过50%）。关于年龄，获得了134只犬和81只猫的数据。牙结石严重程度记录了141只犬和78只猫。
体外培养
口腔拭子送达后立即从运输培养基转移至含有改良Pavlova培养基（Pavlova 1938）（PBS、10%马血清、大米淀粉及青霉素-链霉素；pH 6.8）的无菌Falcon管中，于37º C培养48 h，随后传代至新鲜培养基。每次传代后取一滴培养物用光学显微镜（ZEISS Axioscope）检查，以检测毛滴虫的存在（依据其特征性形态、鞭毛和运动性）。该评估仅用于确认毛滴虫的存在，不用于虫种鉴定。仅在未观察到毛滴虫生长时进行传代。当显微镜检查检出毛滴虫时，即判定该样本为阳性，无需进一步传代。观察到毛滴虫生长的样本保存于− 20 °C以供进一步分析。
DNA提取与扩增
从培养中增殖的毛滴虫（仅来自显示毛滴虫生长的样本）中提取DNA，使用Extractme Genomic DNA KIT（BLIRT S.A，波兰）按照制造商说明进行操作。分离株保存于4°C并在48小时内检查。为确认毛滴虫遗传物质的存在并鉴定虫种，使用T1（5’-GAGAAGTCGTAACAAGGTAACG-3’）和T2（5’-ATGCTTCAGTTCAGCGGGTCT-3’）引物（Genomed，波兰）（Dybicz等，2018）扩增ITS1–5.8S rRNA-ITS2片段。PCR扩增使用Bio-Rad T100™热循环仪进行，PCR条件如下：94 °C初始变性3 min，随后30个循环的94 °C变性30 s、50 °C退火1 min、72 °C延伸1 min，最后72 °C终延伸7 min。PCR产物在2%琼脂糖凝胶中电泳检查，经Midori Green Advance DNA Stain（Nippon Genetics Europe GmbH，Düren，德国）染色后显像。预期产物大小约为370 bp。
DNA测序与数据分析
PCR产物由Genomed（波兰）纯化并进行双向测序。对色谱图进行目视评估以排除双峰的存在。使用BioEdit软件（版本7.2.5.0）进行序列分析。将获得的序列通过blast搜索（https://blast.ncbi.nlm.nih.gov/Blast.cgi）与GenBank中收录的序列进行比较。
统计分析
使用Microsoft Excel和在线统计计算器VassarStats（http://vassarstats.net/）进行统计分析。计算每组的阳性结果比例，并使用Wilson方法估计比例的95%置信区间。变量间关联强度使用比值比（OR）评估，以第一组作为参照类别。当列联表中出现零单元格计数时，应用Haldane–Anscombe校正（+ 0.5）。研究组间毛滴虫感染率的差异使用卡方（χ²）检验评估，显著性水平设定为p ≤ 0.05。在零单元格计数的情况下，使用Fisher精确检验（报告双侧p值），或对于2 × 3表，使用其Freeman–Halton扩展。
结果
口腔毛滴虫在犬中的检出频率显著高于猫（p < 0.05）。体外培养显示，151份犬样本中有26份（17.22%；95% CI，11.75%–24.4%）、87份猫样本中有5份（5.75%；95% CI：2.13%–13.5%）存在毛滴虫。PCR分析证实所有显微镜检查阳性样本中均存在毛滴虫遗传物质。在犬中，患病率最高见于老年动物（9岁以上）和严重牙结石沉积的个体（超过50%的牙齿表面被牙结石覆盖），差异具有统计学显著性。在猫中，年龄、生活环境和口腔卫生状况均无显著差异。详情见表1。
表1. 各组口腔毛滴虫发生的详细数据
在犬中，26份阳性样本中有23份成功测序，最常鉴定的虫种为Trichomonas brixi（65.2%，23份测序样本中的15份），其次为Trichomonas tenax（34.8%，23份测序样本中的8份）。在猫中，测序鉴定出T. tenax（80%，5份样本中的4份）和T. brixi（20%，5份样本中的1份）。本研究报告的核苷酸序列数据已存入GenBank数据库，登录号为PZ658394-PZ658421。详细数据见表2。
表2. 阳性样本中口腔毛滴虫虫种组成的详细数据
讨论
关于食肉动物口腔毛滴虫发生的现有数据有限。在波兰，通过PCR检测确定犬的感染率为4.9%，猫为5.3%（Dybicz等，2018）。本研究中，犬的感染率估计为17.22%，猫为5.75%。尽管猫的结果与Dybicz等的研究相当，但犬的结果明显更高。这可能归因于过去几年流行病学格局的变化。然而，也应考虑到所获结果的差异可能受到诸如使用不同的毛滴虫检测方法和研究组个体饲养条件等因素的影响。在邻国捷克共和国，通过培养法评估的感染率显著高于本研究观察到的结果，犬达37.8%，猫达9.8%（Kellerová和Tachezy，2017）。该研究也包括了收容所动物，但作者认为家养个体比收容所动物感染风险更高。在匈牙利进行的一项基于PCR的研究中，所有检测样本均未检出毛滴虫，尽管研究人群较小可能影响了结果（Tuska-Szalay等，2024）。日本和中国华东地区也使用PCR方法评估了犬的当前流行病学状况。在日本，结果与本研究相当，为14.6%，而在中国人群中发现的阳性个体比例明显较低（2.8%）（Ito等，2023；Gui等，2025）。
在本研究中，犬的口腔毛滴虫检出率显著高于猫（p<0.05）。既往研究也报道了类似结果（Kellerová和Tachezy，2017）。这一差异可能并非由于猫的易感性较低，而是由于行为方式和饲养条件的不同。犬通常有更多机会与其他个体及外部环境接触，这与更高的感染风险相关。此外，猫在与同种个体的互动中通常更为保守，且往往对外部环境的暴露有限，这降低了接触病原体的机会（Menchetti等，2020）。
在本研究中，我们观察到犬口腔毛滴虫的感染率随年龄增长而升高，在9岁以上年龄组达到最高水平。较高的检出频率还与口腔卫生状况差和牙结石严重程度相关。这些因素可能与口腔微环境的变化有关，包括pH改变和低氧可用性部位的形成，这可能促进毛滴虫的定植（Matthew等，2025）。关于年龄，既往研究也报道了类似的观察结果（Kellerová和Tachezy，2017）。值得注意的是，这两个因素可能相互关联，因为犬的口腔卫生水平随年龄增长而下降（Kortegaard等，2008；Hemida等，2025）。有趣的是，本研究中仅在室内猫中检出毛滴虫，尽管室内与室外动物之间的差异无统计学意义。
在本研究中，犬和猫感染了两种毛滴虫：T. tenax和T. brixi，但感染率不同。在犬中，T. brixi比T. tenax更常见（测序样本中分别为65.2%和34.8%），而在猫中观察到相反的模式（80%为T. tenax，20%为T. brixi）。饲养条件似乎再次发挥了重要作用，因为所有检出毛滴虫的猫均为室内动物，与外部环境接触有限但与人类接触更密切。因此，主要感染源似乎是人类，T. tenax被认为是人类的典型虫种。然而，这仅是一种假设，因为未从主人处采集样本，因此该假设无法得到明确证实。T. brixi是一种相对近期描述的虫种，我们的研究首次确认其在波兰的存在。有趣的是，来自捷克共和国和日本的研究也显示它是犬中最常见的毛滴虫（Kellerová和Tachezy，2017；Ito等，2023）。迄今为止，尚未在人类中发现T. brixi，它似乎是犬和猫的典型虫种。三个犬样本未能成功测序。所获序列质量过低（由于缺乏可靠的碱基识别），无法鉴定毛滴虫虫种。序列质量差可能源于DNA数量或完整性不足，以及测序过程中的技术限制。然而，本研究中观察到的测序失败的确切原因无法确定。
值得注意的是，根据现有文献，尽管Pavlova培养基目前并非毛滴虫培养最常用的培养基，但其仍被应用并支持这些原生动物的生长（Tasca et al. 2001; Al-Dabbagh and Kharofa 2007）。Tasca等人（2001）报道，由于可见的淀粉颗粒和细菌生长，该培养基无法对毛滴虫进行准确的显微镜观察。然而，在我们的研究中未遇到类似问题，因为显微镜检查的目的是确定毛滴虫的存在与否，并促进其增殖以用于后续分子分析，而非评估其详细的形态学特征。
总之，本研究展示了波兰犬和猫口腔毛滴虫的当前流行率及物种组成。该研究证实了人兽共患的T. tenax的存在，并首次在波兰的犬和猫中证明了T. brixi的存在。此外，年龄或口腔卫生状况等风险因素在犬中的重要性得到了证实。我们的结果更新和补充了该领域的研究发现，可能对动物和人类健康均具有重要意义。
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The oral cavity provides a suitable environment for various microorganisms, including protozoa such as trichomonads, which may be associated with periodontal disease development. Humans are considered the typical hosts of Trichomonas tenax but it has also been found in dogs, cats, and other animals, which makes it a factor with a significant zoonotic potential. Apart from T. tenax, domestic carnivores may be infected with such species as T. canistomae, T. felistomae and T. brixi. The aim of this study was to determine the prevalence, species composition, and risk factors of trichomonad colonization of the oral cavity in dogs and cats from Poland. Oral swabs from 238 household animals (151 dogs and 87 cats) were examined using cultivation methods. Amplification of ITS1– 5.8 S rRNA-ITS2 fragment was performed for microscopic-positive samples and PCR products were further sequenced. Trichomonads were identified in 26 out of 151 (17.22%) canine and 5 out of 87 (5.75%) feline samples. Higher frequency was observed in older dogs and dogs with intense dental calculus. T. brixi and T. tenax were the only species detected in both dogs and cats. The detection of zoonotic T. tenax in a substantial number of examined animals highlights the relevance of this finding also in the context of human health.
Introduction
Trichomonads are widespread protozoa found in different locations in a broad spectrum of hosts. They have been detected, among others, in the oral cavity of humans and many animal species, including dogs and cats (Dybicz et al. 2018; Nor Azmi et al. 2024; Matthew et al. 2025). The species typical for dogs and cats are Trichomonas canistomae/felistomae or recently described T. brixi, but the zoonotic species T. tenax has also been found (Kellerová and Tachezy 2017). Pathogenicity of oral trichomonads is widely discussed – they are often considered to be non-pathogenic but numerous studies confirm their association with periodontal diseases (Patel et al. 2017; Kadhim et al. 2023; Matthew et al. 2023; Stoyanov et al. 2024). It has been proven that they are capable of producing hemolysins and proteases that damage collagen fibers and promote soft tissue degradation (Nagao et al. 2000; Yamamoto et al. 2000). They have also been detected outside the oral cavity, in the submandibular salivary glands, tonsils, and lymph nodes. Moreover, they have been associated with pulmonary diseases and the formation of abscesses in the lower respiratory tract, particularly in immunocompromised individuals or those with coexisting diseases (Szczepaniak et al. 2016; Kellerová and Tachezy 2017; Dybicz et al. 2018; Duboucher et al. 2000; Hong et al. 2024).
Despite clear evidence of their role as pathogens, trichomonads remain poorly understood in dogs and cats. These domestic animals are particularly important due to their close contact with humans, which increases the risk of transmission of pathogens with a zoonotic potential. The aim of this study was to determine the prevalence and species composition of trichomonads in the oral cavity of dogs and cats from Poland, as well as the influence of animal age, oral hygiene status, and living environment on the pathogen prevalence.
Materials and methods
Materials
The oral swabs were collected between June 2024 and December 2025 from 151 domestic dogs and 87 cats from different regions of Poland (Lower Silesia, Greater Poland, Lodzkie Voivodship, Podlaskie Voivodeship, Pomerania, West Pomeranian Voivodeship, Lubusz Voivodeship, Masovian, Silesia, Subcarpathian) during routine diagnostic and preventive procedures. The study included animals belonging to various breeds. The samples were taken individually from the surface of gingiva and tooth using sterile swabs, placed in the AMIES transport medium (Deltalab, Spain), and delivered to the laboratory at the Division of Parasitology and Invasive Diseases, Wroclaw University of Environmental and Life Sciences within a maximum of 24 h after collection. During transport, the samples were protected from temperature fluctuations by using insulated packaging. The animals were divided into groups according to age (0–4 years old, 5–8 years old, and ≥ 9 years old) and severity of dental calculus (Grade I – lack of dental calculus or up to 25% of the dental surface covered by tartar, Grade II – 25 to 50% of the dental surface covered by tartar, Grade III – over 50% of the dental surface covered by tartar). With regard to age, data were obtained for 134 dogs and 81 cats. The severity of dental calculus was recorded in 141 dogs and 78 cats.
In vitro culture
The oral swabs were immediately transferred after delivery from the transport medium to sterile falcon tubes containing a modified Pavlova medium (Pavlova 1938) (PBS, 10% of horse serum, rice starch, and penicillin – streptomycin; pH 6.8), cultivated for 48 h at 37º C and then passaged to a fresh medium. A drop of each culture was examined after each passage using a light microscope (ZEISS Axioscope) to detect the presence of trichomonads (based on their characteristic shape, flagella, and motility). This assessment was performed only to confirm the presence of trichomonads and was not used for species identification. Passaging was performed only when no trichomonad growth was observed. When trichomonads were detected by microscopic examination, the sample was considered positive and further passages were not required. The samples, in which trichomonad growth was observed were stored at − 20 °C for further analysis.
DNA extraction and amplification
DNA extraction was conducted from trichomonads multiplied in the culture (only from the samples showing trichomonad growth) using the Extractme Genomic DNA KIT (BLIRT S.A, Poland) according to the manufacturer’s instruction. The isolates were stored at 4°C and examined within 48 hours. To confirm the presence of trichomonad genetic material and to identify the species, the ITS1– 5.8S rRNA-ITS2 fragment was amplified using T1 (5’-GAGAAGTCGTAACAAGGTAACG-3’) and T2 (5’-ATGCTTCAGTTCAGCGGGTCT-3’) primers (Genomed, Poland) (Dybicz et al. 2018). PCR amplification was performed using a Bio-Rad T100™ Thermal Cycler, and the PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 72 °C for 7 min. The PCR products were examined electrophoretically in 2% agarose gels and visualized after staining with Midori Green Advance DNA Stain (Nippon Genetics Europe GmbH, Düren, Germany). The expected product size was approximately 370 bp.
DNA sequencing and data analysis
The PCR products were purified and sequenced in both directions by Genomed (Poland). The chromatograms were visually assessed to exclude the presence of double peaks. Sequence analyzing was performed using BioEdit software (version 7.2.5.0). The obtained sequences were compared by a blast search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the sequences deposited in the GenBank.
Statistical analysis
Statistical analyses were performed using Microsoft Excel and the online statistical calculator VassarStats (http://vassarstats.net/). For each group, the proportion of positive results was calculated, and 95% confidence intervals for proportions were estimated using the Wilson method. The strength of the association between variables was assessed using odds ratios (OR), with the first group treated as the reference category. In cases of zero cell counts in contingency tables, the Haldane–Anscombe correction (+ 0.5) was applied. Differences in the prevalence of trichomonads infections between the study groups were evaluated using the chi-square (χ²) test, with a significance level set at p ≤ 0.05. In the case of zero cell counts, Fisher’s exact test was used (reporting a two-tailed p-value), or, for 2 × 3 tables, its Freeman–Halton extension.
Results
Oral trichomonads were detected significantly more frequently in dogs than in cats (p < 0.05). In vitro cultivation showed the presence of trichomonads in 26 out of 151 (17.22%; 95% CI, 11.75%–24.4%) canine and 5 out of 87 (5.75%; 95% CI: 2.13%–13.5%) feline samples. The PCR analysis confirmed the presence of trichomonad genetic material in all microscopically positive samples. In dogs, the highest prevalence was observed in older animals (over 9 years old) and individuals with severe dental tartar deposition (with over 50% of the dental surface covered with calculus), the differences were statistically significant. There were no significant differences associated with age, living environment, and oral hygiene status in cats. Details are presented in Table 1.
Table 1. Detailed data on the occurrence of oral trichomonads in the individual groups
| Species | Group size | Groups | total | No of positive samples | Percent of positive samples (%) | 95% (CI) | OR | p |
|---|---|---|---|---|---|---|---|---|
| Dogs | 134 | Age | ||||||
| 0–4 | 46 | 4 | 8.69 | 2.82–21.69 | p = 0.049* | |||
| 5–8 | 39 | 8 | 20.51 | 9.87–36.94 | 2.71 | |||
| > 9 | 49 | 14 | 28.57 | 17.03–43.47 | 4.20 | |||
| 141 | Severity of the tartar | |||||||
| Grade I | 66 | 5 | 7.58 | 2.82–17.50 | p = 0.0004* | |||
| Grade II | 28 | 4 | 14.29 | 4.68–33.56 | 2.03 | |||
| Grade III | 47 | 17 | 36.17 | 23.06–51.52 | 6.91 | |||
| Cats | 81 | Age | ||||||
| 0–4 | 36 | 3 | 8.33 | 2.18–23.59 | p = 0.5963 | |||
| 5–8 | 25 | 2 | 8.0 | 1.40–27.5 | 1.02 | |||
| > 9 | 20 | 0 | 0 | 0.00-20.04 | 0.23 | |||
| 78 | Severity of the tartar | |||||||
| Grade I | 50 | 3 | 6.0 | 1.56–17.54 | p = 0.4131 | |||
| Grade II | 13 | 0 | 0 | 0.00-28.34 | 0.50 | |||
| Grade III | 15 | 2 | 13.33 | 2.34–41.61 | 2.51 | |||
| 80 | Living environment | |||||||
| outdoor | 12 | 0 | 0 | 0.00-30.12 | p = 0.5935 | |||
| indoor | 68 | 5 | 7.35 | 2.74–17.02 | 2.17 | |||
In dogs, 23 out of 26 positive samples were successfully sequenced, and the most frequently identified species was Trichomonas brixi (65.2%, 15 out of 23 sequenced samples), followed by Trichomonas tenax (34.8%, 8 out of 23 sequenced samples). In cats, the sequencing identified T. tenax (80%, 4 out of 5 samples) and T. brixi (20%, 1 out of 5 samples). Nucleotide sequence data reported in this study have been deposited in the GenBank database with accession numbers PZ658394-PZ658421. Detailed data are provided in Table 2.
Table 2. Detailed data on the species composition of oral trichomonads in positive samples
| Sample ID and GenBank accession number | Closest matching sequences from GenBank | Identity (%) |
|---|---|---|
| Dog18 PZ658394 | Trichomonas brixi KX977526.1 | 100 |
| Dog39 PZ658395 | Trichomonas brixi KX977521.1 | 100 |
| Dog43 PZ658396 | Trichomonas brixi KX977521.1 | 100 |
| Dog52 PZ658397 | Trichomonas brixi KX977521.1 | 100 |
| Dog53 PZ658398 | Trichomonas tenax PX602343.1 | 100 |
| Dog68 PZ658399 | Trichomonas brixi KX977521.1 | 100 |
| Dog75 PZ658400 | Trichomonas brixi KX977521.1 | 100 |
| Dog76 PZ658401 | Trichomonas brixi KX977521.1 | 100 |
| Dog77 PZ658402 | Trichomonas tenax KX977536.1 | 100 |
| Dog80 PZ658403 | Trichomonas brixi LC710808.1 | 99.50 |
| Dog85 PZ658404 | Trichomonas brixi PX602352.1 | 99.69 |
| Dog86 PZ658405 | Trichomonas brixi PX602352.1 | 100 |
| Dog91 PZ658406 | Trichomonas tenax KX186997.1 | 100 |
| Dog93 PZ658407 | Trichomonas brixi KX977526.1 | 100 |
| Dog103 PZ658408 | Trichomonas tenax PX602343.1 | 100 |
| Dog106 PZ658409 | Trichomonas tenax PX602343.1 | 100 |
| Dog110 PZ658410 | Trichomonas tenax KX186997.1 | 100 |
| Dog112 PZ658411 | Trichomonas tenax U86615.1 | 100 |
| Dog1Lodz PZ658412 | Trichomonas tenax KX186997.1 | 100 |
| Dog1M PZ658413 | Trichomonas brixi KX977526.1 | 100 |
| Dog4S PZ658414 | Trichomonas brixi KX977521.1 | 100 |
| Dog17W PZ658415 | Trichomonas brixi KX977521.1 | 100 |
| Dog18W PZ658416 | Trichomonas brixi KX977521.1 | 100 |
| CatK3 PZ658417 | Trichomonas tenax KX186997.1 | 100 |
| CatK8 PZ658418 | Trichomonas tenax U86615.1 | 100 |
| CatK30 PZ658419 | Trichomonas tenax PX602343.1 | 100 |
| CatK31 PZ658420 | Trichomonas tenax PX602345.1 | 100 |
| CatK40 PZ658421 | Trichomonas brixi KX977521.1 | 100 |
Discussion
Available data on the occurrence of oral trichomonads in carnivores are limited. In Poland, the prevalence was determined to be 4.9% in dogs and 5.3% in cats by PCR (Dybicz et al. 2018). In this study, the prevalence was estimated at 17.22% in dogs and 5.75% in cats. Although the result in cats is comparable to that obtained by Dybicz et al., in dogs it is markedly higher. This may be attributed to shifts in the epidemiological landscape over the past several years. However, it should also be taken into account that differences in the obtained results may have been influenced by factors such as the use of different methods for trichomonads detection and the housing conditions of the individuals included in the study group. In the neighboring Czech Republic, the prevalence assessed by the cultivation method was considerably higher than that observed in our study, reaching 37.8% in dogs and 9.8% in cats (Kellerová and Tachezy 2017). That study included also shelter animals, however, the authors suggest that domestic individuals were at a higher risk of infection than the shelter animals. In a PCR-based study conducted in Hungary, no oral trichomonads were detected in any of the examined samples, although the small study population may have affected the results (Tuska-Szalay et al. 2024). Current epidemiological status of dogs was also assessed in Japan and East China, using PCR method. In Japan, the result was comparable to that of our study, amounting to 14.6%, whereas in the Chinese population a markedly lower proportion of positive individuals (2.8%) was found (Ito et al. 2023; Gui et al. 2025).
In our study, oral trichomonads were detected significantly more frequently in dogs than in cats (p<0.05). The previous studies also reported similar results (Kellerová and Tachezy 2017). The difference is likely not due to lower susceptibility in cats, but rather to distinct behaviors and different housing conditions. Dogs generally have greater opportunities for contact with other individuals and the external environment, which is associated with a higher risk of infection. Additionally, cats are typically more reserved in interactions with conspecifics and often have limited exposure to the external environment, which decreases the chance of contact with pathogens (Menchetti et al. 2020).
In the present study, we observed that prevalence of oral trichomonads in dogs increased with age, reaching its highest level in the group of animals older than 9 years. Higher frequency was also associated with poor oral hygiene status and severity of the dental calculus. These factors may be associated with changes in the oral microenvironment, including pH alterations and the development of sites with limited oxygen availability, which could promote colonization by trichomonads (Matthew et al. 2025). Regarding age, similar observations have been reported in previous studies (Kellerová and Tachezy 2017). It is worth noting that these two factors may be associated, since the level of oral hygiene in dogs decreases with age (Kortegaard et al. 2008; Hemida et al. 2025). Interestingly, trichomonads were detected only in indoor cats in this study, although the difference between indoor and outdoor animals was not statistically significant.
In our study, dogs and cats were infected by two species: T. tenax and T. brixi, but at different rate. In dogs, T. brixi was found to be more common than T. tenax (65.2% vs 34.8% of the sequenced samples), whereas an opposite pattern was observed in cats (80% T. tenax vs 20% T. brixi). The housing conditions seem to play a significant role again, as all cats in which oral trichomonads were detected were indoor animals, with limited contact with the external environment but closer contact with humans. Therefore, the primary source of infection seem to be humans, for whom T. tenax is considered a typical species. However, this is only a hypothesis, since samples were not collected from the owners, and therefore this assumption cannot be unequivocally confirmed. T. brixi is a relatively recently described species, and our study confirmed its presence in Poland for the first time. Interestingly, the studies from the Czech Republic and Japan also showed it as the most frequent trichomonad in dogs (Kellerová and Tachezy 2017; Ito et al. 2023). So far, T. brixi has not been found in humans and it seems typical for dogs and cats. Three dog samples could not be successfully sequenced. The obtained sequences were of too low quality (due to the lack of reliable base calls) to enable identification of the trichomonads species. Poor sequence quality may result from insufficient DNA quantity or integrity, as well as from technical limitations during the sequencing process. However, the exact cause of the observed sequencing failure in this study could not be determined.
It is worth noting that, according to the available literature, although Pavlova medium is not currently the most commonly used medium for trichomonad cultivation, it is also applied and supports the growth of these protozoa (Tasca et al. 2001; Al-Dabbagh and Kharofa 2007). Tasca et al. (2001) reported that the medium did not allow for accurate microscopic observation of the trichomonads due to the presence of visible starch granules and bacterial growth. We did not encounter a similar problem in our study, however, the purpose of the microscopic examination was to determine the presence or absence of trichomonads and to facilitate their multiplication for subsequent molecular analyses, rather than to assess their detailed morphological features.
In conclusion, this research showed the current prevalence and species composition of oral trichomonads in dogs and cats from Poland. The study confirmed the presence of the zoonotic T. tenax and demonstrated the presence of T. brixi for the first time in dogs and cats in Poland. Moreover, the significance of risk factors, such as age or oral hygiene status in dogs was confirmed. Our results update and supplement the research findings in this field and may be important for both animal and human health.