兽研摘要
在犬中已描述了毛滴虫门（Parabasalia）的三种不同寄生虫（人五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴）。然而，关于中国实验比格犬中这些毛滴虫物种流行情况的信息有限。本研究的目的是调查这些犬中这三种寄生虫物种的感染频率和分子特征。共从普通清洁级实验比格犬采集452份粪便样本，采用PCR分析进行检测，阳性样本随后进行测序以进行物种鉴定和分析。结果显示，实验犬中人五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴的流行率分别为6.4%（29/452，95% CI：4.1–8.7%）、0%（0/452）和0.7%（3/452，95% CI：0–1.4%）。序列和系统发育分析表明，获得的人五毛滴虫SSU rRNA基因序列与先前报道的CC1基因型相似，脆弱双核阿米巴SSU rRNA基因序列属于基因型1，这是人类中最常见的基因型。本研究首次提供了全球实验比格犬中人五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴分子流行率的数据。
关键词：流行率，人五毛滴虫，胎儿三毛滴虫，脆弱双核阿米巴，实验比格犬，中国
引言
毛滴虫是厌氧、有鞭毛的原生生物，包括致病物种和一些共生物种。某些物种定殖于脊椎动物和无脊椎动物宿主的口腔、消化道和泌尿生殖道，在犬中至少已识别出五个物种：人五毛滴虫（Davaine, 1860）Wenrich, 1931、胎儿三毛滴虫（Riedmüller, 1928）Wenrich & Emmerson, 1933、口腔毛滴虫（Muller, 1773）Dobell, 1939、Brixi毛滴虫 Kellerová & Tachezy, 2017和脆弱双核阿米巴 Jepps & Dobell, 1918 [4, 19, 23, 25]。
人五毛滴虫，原名人毛滴虫 Davaine, 1860，是一种已确认的致病性人兽共患原生动物寄生虫，寄生于人类和其他哺乳动物的盲肠和结肠。它已被认为与人类和各种动物宿主的腹泻有关，以及人类的肺部感染和类风湿性关节炎 [5, 21, 34–35]。胎儿三毛滴虫是一种引人关注的原生动物寄生虫，感染多种动物宿主，主要是牛和猫，偶尔从猪和犬中分离到 [6, 8]。它引起牛毛滴虫病，一种主要导致牛不育和流产的性传播疾病，以及猫毛滴虫病，一种新出现的胃肠道疾病，导致猫慢性大肠性腹泻 [6, 7]。口腔毛滴虫和Brixi毛滴虫是在人类和动物口腔中鉴定出的人兽共患有鞭毛原生动物，与牙周病有关 [19]。脆弱双核阿米巴是全球最常见的人类肠道寄生虫之一，具有阿米巴样形态 [33]。经过形态学和分子研究，该寄生虫在系统发育上被认定为有鞭毛的毛滴虫 [1, 3, 29]。脆弱双核阿米巴已在人类、非人灵长类动物、猪、猫、犬、大鼠、兔、宠物虎皮鹦鹉和节肢动物媒介中检测到，并且人类与动物之间人兽共患传播的可能性已被认识 [9–11, 18, 32–33]。
比格犬是国际公认的标准实验品种，广泛应用于医学、生物学、病理学、肿瘤学和其他生命科学领域 [20]。随着中国生物医学产业的快速发展，对实验比格犬的需求显著增加，国内相关养殖发展迅速。在比格犬的养殖过程中，寄生虫感染不仅对实验比格犬的健康构成严重威胁，而且显著影响科研数据的准确性。迄今为止，尚无关于中国实验比格犬中上述毛滴虫物种存在的流行病学数据。本研究的目的是调查中国实验比格犬中三种毛滴虫物种的流行率和分子特征。
材料与方法
粪便样本采集
2025年3月至5月期间，从北京（150份）、连云港（84份）和南通（218份）的实验比格犬养殖场共采集452份普通清洁级比格犬的新鲜粪便样本。每份粪便标本收集于单独的一次性塑料袋中，在−20 °C保存至DNA提取。由于新鲜粪便样本是从犬笼中收集的（而非直接从动物采集），本研究无需动物伦理批准。
DNA提取和PCR扩增
使用E.Z.N.A® Mag-Bind® Stool DNA Kit（Omega Bio-tek, Norcross, GA, USA）按照制造商说明从每份粪便样本约200 mg中提取基因组DNA。最终核酸在−20 °C保存。
采用表1所列引物对及详细方案，根据Li等（2015）[22]、Vandenberg等（2006）[31]和Cacciò等（2012）[2]先前描述的巢式PCR方法，分别对样本中三种毛滴虫的特定SSU rRNA基因区域进行巢式PCR扩增。第一轮PCR以提取的粪便基因组DNA为模板，第二轮PCR以第一轮PCR产物为模板。所有PCR分析均包含阳性和阴性对照（蒸馏水）。第二轮PCR产物在1.5%琼脂糖凝胶上电泳，并使用凝胶成像系统观察。病原体阳性样本的扩增产物用相应扩增引物进行双向测序（生工生物工程（上海）股份有限公司，中国上海）。
Table 1. Primers and PCR procedure for three trichomonad species in this study.
| Primers | Sequences (5′–3′) | PCR procedure | Size (bp) | Reference | |
|---|---|---|---|---|---|
| First PCR for P. hominis and T. foetus | FF | GCGCCTGAGAGATAGCGACTA | 95 °C for 10 min, then 30 cycles (95 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s), followed by 72 °C for 10 min | [ 22 ] | |
| RR | GGACCTGTTATTGCTACCCTCTTC | ||||
| Second PCR for P. hominis | hF | TGTAAACGATGCCGACAGAG | 95 °C for 10 min, then 30 cycles (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s), followed by 72 °C for 10 min | 339 | |
| hR | CAACACTGAAGCCAATGCGAGG | ||||
| Second PCR for T. foetus | sF | GGTTGTTTGTATAGGATTGC | 95 °C for 10 min, then 30 cycles (95 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s), followed by 72 °C for 10 min | 452 | |
| sR | TGCCCTCATAAAAGGACAA | ||||
| First PCR for D. fragilis | DF1 | CTCATAATCTACTTGGAACCAATT | 95 °C for 4 min, followed by 40 cycles, each consisting of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min | [ 31 ] | |
| DF4 | CCCCGATTATTCTCTTTGATATT | ||||
| Second PCR for D. fragilis | DF322For | GAGAAGGCGCCTGAGAGATA | 95 °C for 4 min, followed by 40 cycles, each consisting of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min | 366 | [ 2 ] |
| DF687Rev | TTCATACTGCGCTAAATCATT | ||||
序列与系统发育分析
本研究获得的合格正向和反向原始序列使用DNAMAN 6.0软件中的de novo组装插件组装为最终一致序列。最终序列用BioEdit7.1（http://www.mbio.ncsu.edu/BioEdit/bioedit.html）比对，并通过BLAST检索（http://www.ncbi.nlm.nih.gov/BLAST）进行检查。系统发育采用MEGA 6.06（http://www.megasoftware.net/）中的邻接法（NJ）以Kimura 2参数模型推断，并进行1,000次自举重复。本研究测序的代表性SSU rRNA已提交至GenBank，人五毛滴虫的登录号为PZ028360–PZ028361，脆弱双核阿米巴的登录号为PZ028364。
结果
人五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴的感染频率
特异性PCR在452份样本中分别检出人五毛滴虫29份、胎儿三毛滴虫0份和脆弱双核阿米巴7份。Sanger测序确认全部29份PCR阳性样本中均存在人五毛滴虫，但仅3份脆弱双核阿米巴PCR阳性样本测序成功。仅这些经证实的样本被视为阳性（表2）。据此，通过PCR分析确定样本中人五毛滴虫和脆弱双核阿米巴的总感染率分别为6.4%（29/452，95% CI：4.1–8.7%）和0.7%（3/452，95% CI：0–1.4%）（表2）。本研究中未检出胎儿三毛滴虫感染。其余4份脆弱双核阿米巴PCR阳性样本测序失败，因此被归类为阴性。
Table 2. Prevalence of P. hominis, T. foetus and D. fragilis infections in experimental Beagle dogs in China.
| Sampling site | No. samples | P. hominis | T. foetus | D. fragilis | |||
|---|---|---|---|---|---|---|---|
| No. positive | Positive rate/% (95% CI) | No. positive | Positive rate/% (95% CI) | No. positive | Positive rate/% (95% CI) | ||
| Beijing | 150 | 6 | 4.0 (0.8–7.2) | 0 | 0 | 1 | 0.7 (0–2.0) |
| Lian Yun Gang | 84 | 2 | 2.4 (0–5.7) | 0 | 0 | 0 | 0 |
| Nan Tong | 218 | 21 | 9.6 (5.7–13.6) | 0 | 0 | 2 | 0.9 (0–2.2) |
| Total | 452 | 29 | 6.4 (4.1–8.7) | 0 | 0 | 3 | 0.7 (0–1.4) |
北京、连云港和南通比格犬中人五毛滴虫的感染率分别为4.0%（6/150，95% CI：0.8–7.2%）、2.4%（2/84，95% CI：0–5.7%）和9.6%（21/218，95% CI：5.7–13.6%）。北京、连云港和南通比格犬中脆弱双核阿米巴的感染率分别为0.7%（1/150，95% CI：0–2.0%）、0.0%（0/84）和0.9%（2/218，95% CI：0–2.2%）。本研究中未发现人五毛滴虫和脆弱双核阿米巴的混合感染。
人五毛滴虫和脆弱双核阿米巴的分子分析
29份人五毛滴虫阳性PCR扩增产物测序成功，本研究获得两种不同序列（Seq1，GenBank登录号：PZ028360，n = 26；Seq2，GenBank登录号：PZ028361，n = 3），与先前报道的基因型CC1（GenBank登录号：KJ408929）相比，这两种序列分别存在1个和2个核苷酸差异[24]。基于NJ法的系统发育分析也显示，本研究获得的两种序列与来自几种动物宿主的人五毛滴虫分离株形成单系簇（图1）。
[图：邻接树显示，从实验比格犬获得的人五毛滴虫SSU rRNA序列]
在总共7份D. fragilis PCR阳性样本中，仅有3份获得了成功的测序结果。三株分离株序列相同，代表单一分离株（GenBank登录号：KJ408929）。Blast分析显示其与来自人类和虎皮鹦鹉的若干分离株完全一致。NJ树显示，本研究获得的序列与来自人类和虎皮鹦鹉的基因型1分离株形成单系簇（图2）。
[图：不同毛滴虫分离株SSU rRNA序列的系统发育分析。Dientamoeba fragilis分离株重新]
讨论
在实验动物中，研究人员更关注动物在科学研究中的使用，而非动物自身的疾病。关于实验比格犬中人兽共患病原体存在情况的知识有限，据我们所知，目前全球尚无关于实验比格犬中毛滴虫种类的报道。
五毛滴虫已在世界范围内报道，可感染多种动物宿主，尤其在猪、牛、山羊和犬中流行率较高[26, 34]。本研究中实验比格犬的五毛滴虫流行率为6.4%（29/452），低于中国北方（27.4%，69/252）、华东（31.4%，99/315）和华南（22.4%，66/294）地区报道的犬五毛滴虫感染率[17, 23–24]。我们观察到的水平也低于法国（15.8%，34/215）、波兰（12.2%，5/41）和美国（92.9%，13/14）的发现，而我们的结果与日本（7.0%，38/544）的发现相似[13, 16, 27, 30]。
胎儿三毛滴虫是一种原生动物寄生虫，传统上被认为是牛生殖道疾病和猫胃肠道感染的病因[6]。近年来，胎儿三毛滴虫或类胎儿三毛滴虫已相继从肺炎或急性呼吸综合征患者体内分离到，提示胎儿三毛滴虫可能具有一定的人兽共患潜力[25]。自Gookin等人于2005年[12]首次报道犬可感染胎儿三毛滴虫以来，分子方法表明犬胎儿三毛滴虫感染率在美国为5.3%（2/38）、华东为0.6%（2/315）、华南为5.8%（17/294）[17, 24, 30]。然而，本研究中未检测到胎儿三毛滴虫感染。
脆弱双核阿米巴是人类和动物研究中被忽视的毛滴虫，尽管自其首次发现以来已超过100年[4]。除人类数据外，关于脆弱双核阿米巴自然宿主范围的信息很少。已发表的零星数据表明，脆弱双核阿米巴已在少数动物物种中检测到，包括大鼠、猫、犬、牛、猪、山羊、兔、绵羊、豚鼠、虎皮鹦鹉和非人灵长类动物（表3）。对于犬，仅可使用最近发表的两篇报道的qPCR数据，感染率在澳大利亚为1.8%（1/56），在捷克为7.4%（4/54）[4, 18]。基于PCR产物的Sanger测序，本研究发现中国普通清洁级实验比格犬中脆弱双核阿米巴的分子流行率为0.7%（3/452）。
Table 3. Molecular detection of D. fragilis in non-human hosts and foods in various studies.
| Host | Country | Detection method | Positive rate/% | Reference |
|---|---|---|---|---|
| Gorilla | Australia | PCR | 30.0 (3/10) | [ 28 ] |
| Pig | Italy | qPCR, PCR and sequencing | 63.2 (24/38) | [ 2 ] |
| Dog | Australia | qPCR | 1.8 (1/56) | [ 4 ] |
| Cat | qPCR | 2.3 (1/43) | ||
| Cattle | Türkiye | PCR | 19.0 (31/163) | [ 33 ] |
| Budgerigar | Türkiye | qPCR | 21.3 (32/150) | [ 32 ] |
| Dog | Czechia | qPCR | 7.4 (4/54) | [ 18 ] |
| Cat | qPCR | 5.3 (1/19) | ||
| Horse | qPCR | 13.3 (2/15) | ||
| Rabbit | qPCR | 38.5 (5/13) | ||
| Sheep | qPCR | 16.7 (1/6) | ||
| Goat | qPCR | 25.0 (1/4) | ||
| Guinea pig | qPCR | 50.0 (1/2) | ||
| Rabbit | PCR | 7.7 (1/13) | ||
| Housefly | Türkiye | PCR | 8.0 (12/150) | [ 10 ] |
| Cattle | Australia | qPCR | 73.5 (36/49) | [ 14 ] |
| Raw Milk and Cheese | Türkiye | PCR | 6.0 (9/150) | [ 11 ] |
与宠物犬、家养犬和农场犬相比，实验犬的饲养有严格的环境和管理要求，本研究中犬的五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴感染率较低，可能很大程度上归因于此。
本研究获得的五毛滴虫SSU rRNA基因序列与先前报道的五毛滴虫基因型CC1相比，表现出微小的等位基因变异。这可能涉及任何给定基因组中SSU rRNA基因多拷贝内的预期变异，也可能源于寄生虫-宿主或寄生虫-环境的适应[23]。目前，分子分析已证明脆弱双核阿米巴的遗传变异性有限，已描述两种基因型，命名为基因型1和基因型2，其中基因型1已在广泛的宿主中报道，包括人类和动物[2]。SSU rRNA部分基因区域分析显示，本研究获得的序列属于基因型1，与来自人类和虎皮鹦鹉的若干分离株具有100%一致性，提示存在人兽共患传播的潜力。
已发表的数据，包括从犬中分离出人五毛滴虫和胎儿三毛滴虫菌株以及动物感染实验，支持犬是人五毛滴虫和胎儿三毛滴虫的天然宿主[12, 21]。然而，基于以往和本研究，很难得出犬可作为脆弱双核阿米巴天然宿主的结论[4, 14]。以往的报道仅提供了犬中脆弱双核阿米巴的qPCR数据，而这些检测方法产生的qPCR结果需要优化和验证以确认其准确性，因为有报道显示qPCR检测可能与非目标物种发生交叉反应[4, 14]。这三只比格犬是自然感染了脆弱双核阿米巴，还是仅仅通过脆弱双核阿米巴包囊或前包囊阶段的机械性传代而非本土感染，仍不确定。此外，本研究中无临床症状、粪便中未检出滋养体或包囊、以及PCR检测率相对较低，进一步表明得出比格犬是脆弱双核阿米巴天然宿主的结论为时过早。
目前，这三种毛滴虫物种的许多特征，尤其是脆弱双核阿米巴，包括该寄生虫的生物学、生活史、宿主范围、致病性、可能的传播途径和遗传多样性等，尽管近年来取得了一些重要进展，但充其量仍知之甚少。据我们所知，本研究是中国首次对实验比格犬中人五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴进行的分子评估。然而，本研究存在一些局限性。首先，样本量和采样区域相对有限，仅涉及三个城市和452份样本。其次，未同时采集与实验比格犬有密切接触的工作人员的粪便样本以及环境样本，并对其进行这三种毛滴虫物种的检测，这使得难以评估这些毛滴虫物种的任何潜在人兽共患传播。最后，几种已发表的脆弱双核阿米巴分子检测方法，包括本研究采用的方法，在动物粪便标本中会与其他密切相关的毛滴虫如胎儿三毛滴虫、人五毛滴虫和移动三毛滴虫发生交叉反应[4, 14]。这意味着虽然这些分子方法可用于动物样本中脆弱双核阿米巴的检测，但需要包括显微镜检查或测序在内的额外证据来确认新宿主物种中的脆弱双核阿米巴感染[15]。然而，本研究仅使用PCR和测序来分析脆弱双核阿米巴，未应用粪便显微镜检查。这也是在计算观察到的感染率时，其余四份未能测序的脆弱双核阿米巴PCR阳性样本未被认定为阳性的原因。这凸显了迫切需要利用大规模采样和不同动物物种来阐明这些毛滴虫物种的人兽共患传播，并开发一种快速、灵敏的分子检测方法用于动物样本中毛滴虫物种尤其是脆弱双核阿米巴的检测。
结论
我们的研究结果提供了关于清洁级实验比格犬中人五毛滴虫、胎儿三毛滴虫和脆弱双核阿米巴相关潜在人兽共患风险的认识，并强调需要通过全面的病原检测和综合感染控制来改善实验动物的健康和微生物学质量。
致谢
我们感谢养殖场工作人员对本研究的协助。
本文引用格式：Liao X-L, Ge X-L, Gong R-J, Ma J-M, Wang Y-H, Gui L-Q, Guo Q-H, Liu X-C & Li W-C. 2026. First detection and molecular characterization of three trichomonad species in experimental beagle dogs in China. Parasite 33, 56. https://doi.org/10.1051/parasite/2026056.
编辑：Jean-Lou Justine。
基金
本工作得到安徽科技学院校级人才项目（编号：dkwd201702，负责人：W. Li）和安徽科技学院兽医科学高峰学科项目（编号：XK-XJGF002，负责人：W. Li）的资助。
利益冲突
所有作者声明无竞争性利益。
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Abstract
Three different parasites of the phylum Parabasalia (Pentatrichomonas hominis, Tritrichomonas foetus and Dientamoeba fragilis) have been described in dogs. However, limited information about the prevalence of these trichomonad species in experimental beagle dogs in China is available. The aim of the current study was to investigate the frequency and molecular characterization of these three parasites species in these dogs. A total of 452 fecal samples were collected from ordinary clean-grade experimental beagle dogs and examined using PCR analysis, and positive samples were subsequently sequenced for species identification and analysis. Results showed that the prevalence of P. hominis, T. foetus and D. fragilis in the experimental dogs was 6.4% (29/452, 95% CI: 4.1–8.7%), 0% (0/452) and 0.7% (3/452, 95% CI: 0–1.4%), respectively. The sequence and phylogenetic analyses showed that the obtained sequences of P. hominis SSU rRNA genes were similar to the previously reported genotype CC1, and the sequences of D. fragilis SSU rRNA genes belonged to genotype 1, which is the most common genotype in humans. Our study provides the first data on the molecular prevalence of P. hominis, T. foetus and D. fragilis in experimental beagle dogs globally.
Keywords: Prevalence, Pentatrichomonas hominis, Tritrichomonas foetus, Dientamoeba fragilis, Experimental beagle dogs, China
Introduction
Trichomonads are anaerobic, flagellated protists, including both pathogenic species and some symbiotic species. Certain species colonize the oral, digestive and urogenital tracts of vertebrate and invertebrate hosts, with at least five species recognized in dogs: Pentatrichomonas hominis (Davaine, 1860) Wenrich, 1931, Tritrichomonas foetus (Riedmüller, 1928) Wenrich & Emmerson, 1933, Trichomonas tenax (Muller, 1773) Dobell, 1939, Trichomonas brixi Kellerová & Tachezy, 2017 and Dientamoeba fragilis Jepps & Dobell, 1918 [4, 19, 23, 25].
Pentatrichomonas hominis, formerly known as Trichomonas hominis Davaine, 1860, is an identified pathogenic zoonotic protozoan parasitizing the cecum and colon of humans and other mammals. It has been associated with diarrhea in humans and various animal hosts, as well as pulmonary infections and rheumatoid arthritis in human [5, 21, 34–35]. Tritrichomonas foetus is an intriguing protozoan parasite that infects multiple animal hosts, primarily cattle and cats, with occasional isolation from pigs and dogs [6, 8]. It causes bovine trichomoniasis, a sexually transmitted disease mainly leading to infertility and abortion in cattle, and feline trichomoniasis, a newly emerging gastrointestinal disorder that results in chronic large bowel diarrhea in felines [6, 7]. Trichomonas tenax and Trichomonas brixi are zoonotic flagellated protozoans identified in the oral cavities of humans and animals and have been associated with periodontal disease [19]. Dientamoeba fragilis is one of the most common parasites of the human bowel worldwide, with amoeba-like morphology [33]. Following morphological and molecular studies, this parasite was recognized phylogenetically as a flagellated trichomonad [1, 3, 29]. Dientamoeba fragilis has been detected in humans, non-human primates, pigs, cats, dogs, rats, rabbits, pet budgerigars, and arthropod vectors, and the possibility of zoonotic transmission between humans and animals has been recognized [9–11, 18, 32–33].
Beagle dogs are an internationally recognized standard experimental breed widely used in medicine, biology, pathology, oncology and other life sciences [20]. With the rapid development of China’s biomedical industry, the demand for experimental beagles has increased significantly, and domestic related breeding has developed rapidly. During the breeding process of beagle dogs, parasitic infections not only pose a serious threat to the health of experimental beagles, but also significantly affect the accuracy of scientific research data. So far, no epidemiologic data regarding the presence of the above mentioned trichomonad species in experimental beagle dogs in China are available. The aim of this study was to investigate the prevalence and molecular characterization of three trichomonad species in experimental beagle dogs in China.
Materials and methods
Collection of fecal samples
A total of 452 fresh fecal samples of ordinary clean-grade beagle dogs were collected from experimental beagle dog farms in Beijing (150), Lianyungang (84) and Nantong (218), between March to May 2025. Each fecal specimen was collected in an individual disposable plastic pouch, and stored at −20 °C until DNA extraction. Because fresh fecal droppings were collected from the cages of dogs (not from the animals directly), no animal ethics approval was required in this study.
DNA extraction and PCR amplification
Genomic DNA was extracted from approximately 200 mg of each fecal sample with an E.Z.N.A® Mag-Bind® Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA), according to the manufacturer’s instructions. The final nucleic acids were stored at −20 °C.
Three trichomonad species in samples were amplified by nested PCR assays of specific SSU rRNA gene regions using the primer pairs and detailed protocols listed in Table 1, according to the nested PCRs previously described by Li et al., 2015 [22], Vandenberg et al., 2006 [31], and Cacciò et al., 2012 [2], respectively. The first round of PCR used the extracted fecal genomic DNA as a template, and the second round of PCR used the first round of PCR product as a template. All the PCR analyses contained positive and negative controls (distilled water). The secondary PCR products were electrophoresed on a 1.5% agarose gel and visualized using a gel documentation system. Amplicons from pathogen-positive samples were sequenced in both directions with corresponding amplification primers (Sangon Biotech, Shanghai, China).
Table 1. Primers and PCR procedure for three trichomonad species in this study.
| Primers | Sequences (5′–3′) | PCR procedure | Size (bp) | Reference | |
|---|---|---|---|---|---|
| First PCR for P. hominis and T. foetus | FF | GCGCCTGAGAGATAGCGACTA | 95 °C for 10 min, then 30 cycles (95 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s), followed by 72 °C for 10 min | [ 22 ] | |
| RR | GGACCTGTTATTGCTACCCTCTTC | ||||
| Second PCR for P. hominis | hF | TGTAAACGATGCCGACAGAG | 95 °C for 10 min, then 30 cycles (95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s), followed by 72 °C for 10 min | 339 | |
| hR | CAACACTGAAGCCAATGCGAGG | ||||
| Second PCR for T. foetus | sF | GGTTGTTTGTATAGGATTGC | 95 °C for 10 min, then 30 cycles (95 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s), followed by 72 °C for 10 min | 452 | |
| sR | TGCCCTCATAAAAGGACAA | ||||
| First PCR for D. fragilis | DF1 | CTCATAATCTACTTGGAACCAATT | 95 °C for 4 min, followed by 40 cycles, each consisting of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min | [ 31 ] | |
| DF4 | CCCCGATTATTCTCTTTGATATT | ||||
| Second PCR for D. fragilis | DF322For | GAGAAGGCGCCTGAGAGATA | 95 °C for 4 min, followed by 40 cycles, each consisting of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min | 366 | [ 2 ] |
| DF687Rev | TTCATACTGCGCTAAATCATT | ||||
Sequence and phylogenetic analyses
The qualified forward and reverse raw sequences obtained in this study were assembled into a final consensus sequence using the de novo assembly plugin in DNAMAN 6.0 software. Final sequences were aligned with BioEdit7.1 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and examined with BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST). The phylogeny was inferred using a neighbor-joining (NJ) method with the Kimura 2-parameter model in MEGA 6.06 (http://www.megasoftware.net/), with 1,000 bootstrap replicates. Representative SSU rRNAs sequenced in this study have been deposited in GenBank under accession numbers PZ028360–PZ028361 for P. hominis and PZ028364 for D. fragilis.
Results
Frequency of P. hominis, T. foetus and D. fragilis
The specific PCR detected P. hominis, T. foetus and D. fragilis in 29, 0 and 7 samples out of 452 samples. Sanger sequencing confirmed the presence of P. hominis in all 29 PCR positive samples, but only 3 D. fragilis PCR positive samples were sequenced successfully. Only these verified samples were considered positive (Table 2). According to this, the overall prevalence of P. hominis and D. fragilis in the samples, as determined by PCR analysis, was 6.4% (29/452, 95% CI: 4.1–8.7%) and 0.7% (3/452, 95% CI: 0–1.4%), respectively (Table 2). No T. foetus infections were detected in this study. The remaining four D. fragilis PCR positive samples failed to sequence, so they were classified as negative.
Table 2. Prevalence of P. hominis, T. foetus and D. fragilis infections in experimental Beagle dogs in China.
| Sampling site | No. samples | P. hominis | T. foetus | D. fragilis | |||
|---|---|---|---|---|---|---|---|
| No. positive | Positive rate/% (95% CI) | No. positive | Positive rate/% (95% CI) | No. positive | Positive rate/% (95% CI) | ||
| Beijing | 150 | 6 | 4.0 (0.8–7.2) | 0 | 0 | 1 | 0.7 (0–2.0) |
| Lian Yun Gang | 84 | 2 | 2.4 (0–5.7) | 0 | 0 | 0 | 0 |
| Nan Tong | 218 | 21 | 9.6 (5.7–13.6) | 0 | 0 | 2 | 0.9 (0–2.2) |
| Total | 452 | 29 | 6.4 (4.1–8.7) | 0 | 0 | 3 | 0.7 (0–1.4) |
The prevalence rates of P. hominis in beagle dogs in Beijing, Lianyungang and Nantong were 4.0% (6/150, 95% CI: 0.8–7.2%), 2.4% (2/84, 95% CI: 0–5.7%) and 9.6% (21/218, 95% CI: 5.7–13.6%), respectively. The prevalence rates of D. fragilis in beagle dogs in Beijing, Lianyungang and Nantong were 0.7% (1/150, 95% CI: 0–2.0%), 0.0% (0/84) and 0.9% (2/218, 95% CI: 0–2.2%), respectively. There were no coinfections with P. hominis and D. fragilis in the present study.
Molecular analysis of P. hominis and D. fragilis
The 29 P. hominis-positive PCR amplicons were sequenced successfully, and two different sequences were obtained in the present study (Seq1, GenBank accession: PZ028360, n = 26; Seq2, GenBank accession: PZ028361, n = 3), and the two sequences differed at 1 and 2 nucleotides, compared with the previously reported genotypes CC1 (GenBank accession: KJ408929), respectively [24]. Phylogenetic analyses based on the NJ method also showed that the two sequences obtained in this study formed a monophyletic cluster with the P. hominis isolates from several animal hosts (Fig. 1).
[图：Neighbor-joining trees showing that the SSU rRNA sequences of Pentatrichomonas hominis obtained from the experimental be]
Of a total of 7 D. fragilis PCR-positive samples, only 3 yielded successful sequencing results. Three isolates had identical sequences and represented a single isolate (GenBank accession: KJ408929). Blast analyses displayed whole identity with several isolates reported from humans and budgerigars. The NJ tree showed that the sequence obtained in this study formed a monophyletic cluster with the isolates of genotype 1 from humans and budgerigars (Fig. 2).
[图：Phylogenetic analysis of the SSU rRNA sequences from different trichomonad isolates. The Dientamoeba fragilis isolate re]
Discussion
In experimental animals, researchers pay more attention to the use of the animals in scientific research than to the diseases of the animals themselves. Knowledge on the presence of zoonotic pathogens in experimental beagles is limited, and as far as we know, there are currently no reports on trichomonad species in experimental beagles globally.
Pentatrichomonas hominis has been reported worldwide and can infect many animal hosts, especially with a high prevalence in pigs, cattle, goats, and dogs [26, 34]. The prevalence of P. hominis in experimental beagles in this study is 6.4% (29/452), which was lower than the reported rates of canine P. hominis in the northern (27.4%, 69/252), eastern (31.4%, 99/315) and southern (22.4%, 66/294) regions of China [17, 23–24]. Our observed levels are also lower than those found in France (15.8%, 34/215), Poland (12.2%, 5/41), and the United States (92.9%, 13/14), while our results are similar to those found in Japan (7.0%, 38/544) [13, 16, 27, 30].
Tritrichomonas foetus is a protozoan parasite that has been traditionally identified as a cause of reproductive tract disease in cattle and gastrointestinal tract infection in cats [6]. In recent years, T. foetus or T. foetus-like trichomonads have successively been isolated from the bodies of patients with pneumonia or acute respiratory syndrome, suggesting that T. foetus may possess a certain potential for zoonosis [25]. Since Gookin et al., 2005 [12] first reported that dogs can be infected with T. foetus, molecular methods have indicated that the infection rate of T. foetus in dogs is 5.3% (2/38) in the United States, 0.6% (2/315) in eastern China and 5.8% (17/294) in southern China [17, 24, 30]. However, no T. foetus infections were detected in this study.
Dientamoeba fragilis is a neglected trichomonad in human and animal studies, even though it has been more than 100 years since its initial discovery [4]. Aside from data in humans, little information is available about the natural host range of D. fragilis. Sparse published data indicate that D. fragilis has been detected in a few animal species including rats, cats, dogs, cows, pigs, goats, rabbits, sheep, guinea pigs, budgerigars and non-human primates (Table 3). For dogs, only qPCR data from two recently published reports can be used, with infection rates of 1.8% (1/56) in Australia and 7.4% (4/54) in Czechia [4, 18]. Based on Sanger sequencing of the PCR products, the molecular prevalence of D. fragilis in ordinary clean-grade experimental beagle dogs in China was found to be 0.7% (3/452) in the present study.
Table 3. Molecular detection of D. fragilis in non-human hosts and foods in various studies.
| Host | Country | Detection method | Positive rate/% | Reference |
|---|---|---|---|---|
| Gorilla | Australia | PCR | 30.0 (3/10) | [ 28 ] |
| Pig | Italy | qPCR, PCR and sequencing | 63.2 (24/38) | [ 2 ] |
| Dog | Australia | qPCR | 1.8 (1/56) | [ 4 ] |
| Cat | qPCR | 2.3 (1/43) | ||
| Cattle | Türkiye | PCR | 19.0 (31/163) | [ 33 ] |
| Budgerigar | Türkiye | qPCR | 21.3 (32/150) | [ 32 ] |
| Dog | Czechia | qPCR | 7.4 (4/54) | [ 18 ] |
| Cat | qPCR | 5.3 (1/19) | ||
| Horse | qPCR | 13.3 (2/15) | ||
| Rabbit | qPCR | 38.5 (5/13) | ||
| Sheep | qPCR | 16.7 (1/6) | ||
| Goat | qPCR | 25.0 (1/4) | ||
| Guinea pig | qPCR | 50.0 (1/2) | ||
| Rabbit | PCR | 7.7 (1/13) | ||
| Housefly | Türkiye | PCR | 8.0 (12/150) | [ 10 ] |
| Cattle | Australia | qPCR | 73.5 (36/49) | [ 14 ] |
| Raw Milk and Cheese | Türkiye | PCR | 6.0 (9/150) | [ 11 ] |
Compared to pet dogs, household dogs and farm dogs, the breeding of experimental dogs has strict environmental and management requirements, and the low infection rates by P. hominis, T. foetus and D. fragilis in the dogs in this study may be largely attributed to this.
The obtained sequences of the P. hominis SSU rRNA genes in this study exhibit minor allelic variations compared with the previously reported genotypes CC1 of P. hominis. This may involve expected variation within the multiple copies of the SSU rRNA genes in any given genome or may result from parasite-host or parasite-environment adaptation [23]. Currently, molecular analysis has demonstrated limited genetic variability within D. fragilis, and two genotypes, designated genotype 1 and genotype 2, have been described, with genotype 1 reported in a wide range of hosts, including both humans and animals [2]. Analysis of the SSU rRNA partial gene region showed that the sequence obtained in this study belongs to genotype 1 and has 100% identity with several isolates reported from humans and budgerigars, suggesting the potential for zoonotic transmission.
The published data, including the isolation of P. hominis and T. foetus strains from dogs and animal infection, have supported dogs as natural hosts for P. hominis and T. foetus [12, 21]. However, it is very difficult to draw the conclusion that dogs can act as a natural host of D. fragilis based on the previous and present research [4, 14]. The previous reports provided only qPCR data for D. fragilis in dogs, and the qPCR results generated by these assays need to be optimized and validated to confirm their accuracy, because reports have revealed that qPCR assays may cross-react with non-target species [4, 14]. Whether the three beagle dogs were infected naturally with D. fragilis or merely via mechanical passage of the cyst or pre-cyst stages of D. fragilis rather than indigenous infections remains uncertain. Moreover, the absence of clinical signs, the absence of trophozoites or cyst detection in feces, and the relatively low detection rate by PCR in this study further indicate that it is premature to conclude that beagle dogs are natural hosts to D. fragilis.
Currently, many characteristics of the three trichomonad species, especially D. fragilis, including the parasite’s biology, life cycle, host range, pathogenicity, possible transmission routes and genetic diversity etc., remain poorly understood at best, although some important advances have been made in recent years. The present study was, to our knowledge, the first molecular assessment of P. hominis, T. foetus and D. fragilis in experimental beagle dogs in China. However, this study has some limitations. First, the sample size and sampling regions are relatively limited, involving only three cities and 452 samples. Second, fecal samples from staff who have been in close contact with the experimental beagle dogs, as well as environmental samples, were not collected simultaneously and tested for the three trichomonad species, which makes it difficult to assess any potential zoonotic transmission of these trichomonad species. Finally, several published D. fragilis molecular assays, including the method employed in this study, cross react with other closely related trichomonads such as T. foetus, P. hominis and T. mobilensis, in animal stool specimens [4, 14]. This means that while these molecular methods can be used for the detection of D. fragilis in animal samples, additional evidence including microscopy or sequencing are required to confirm D. fragilis infection in a new host species [15]. However, the present study used only PCR and sequencing to analyse D. fragilis, and fecal microscopy was not applied here. This is also the reason why the remaining four D. fragilis PCR-positive samples that failed to be sequenced were not recognized as positive when calculating the observed infection rate. This highlights the urgent need to clarify zoonotic transmission of these trichomonad species by using large-scale sampling and different animal species, and to develop a rapid and sensitive molecular assay for trichomonad species, especially D. fragilis, in animal samples.
Conclusion
Our findings provide insights into the potential zoonotic risks associated with P. hominis, T. foetus and D. fragilis in clean-grade experimental beagle dogs and highlight the need to improve the health and microbiological quality of experimental animals through comprehensive pathogen detection and integrated infection control.
Acknowledgments
We thank the staff on the farms for their assistance with this study.
Cite this article as: Liao X-L, Ge X-L, Gong R-J, Ma J-M, Wang Y-H, Gui L-Q, Guo Q-H, Liu X-C & Li W-C. 2026. First detection and molecular characterization of three trichomonad species in experimental beagle dogs in China. Parasite 33, 56. https://doi.org/10.1051/parasite/2026056.
Edited by Jean-Lou Justine.
Funding
This work was supported by the school-level talent project of Anhui Science and Technology University (No. dkwd201702 to W. Li), Veterinary Science Peak Discipline Project of Anhui Science and Technology University (No. XK-XJGF002 to W. Li).
Conflicts of interest
All authors declare that they have no competing interests.
References
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