曼谷寺庙流浪猫中十二指肠贾第虫（Giardia duodenalis）的分子检测与遗传特征分析

《Frontiers in Parasitology》：Molecular detection and genetic characterization of Giardia duodenalis in temple stray cats in Bangkok, Thailand

　　

摘要专业翻译 引言： Giardia duodenalis（十二指肠贾第虫）是一种普遍存在的肠道原虫，可感染全球范围内的人类、家养动物及野生动物。在泰国，高密度的流浪动物构成了重要的管理挑战。然而，关于流浪猫的分子流行病学数据仍然有限。本研究评估了泰国曼谷寺庙流浪猫中 G. duodenalis（十二指肠贾第虫）的感染率，鉴定了所发现的基因聚合群（genetic assemblages），并探讨了与聚合酶链式反应（PCR）阳性相关的因素。 方法： 研究人员从曼谷24个区的43座寺庙中收集了500只流浪猫的粪便样本。采用针对小亚基核糖体RNA（SSU rRNA）基因的巢式PCR进行 G. duodenalis（十二指肠贾第虫）检测。阳性样本进一步通过谷氨酸脱氢酶（gdh）和磷酸丙糖异构酶（tpi）位点进行基因分型。基于Tamura 3参数模型，采用最大似然法进行系统发育分析。使用多变量逻辑回归评估相关因素。 结果： G. duodenalis（十二指肠贾第虫）的总体感染率为10.8%（54/500；95% CI: 8.4–13.8%），阳性病例分布于24个被调查区中的12个区和43个被采样寺庙中的19个。成功对8个分离株进行基因分型，鉴定出聚合群B（亚聚合群BIV，n = 4）、聚合群F（n = 3）和聚合群A（亚聚合群AI，n = 1）。缺乏人类接触被确定为与较高PCR阳性几率相关的探索性变量（调整后优势比 [aOR] = 8.80；95% CI: 2.15–35.98；P = 0.002），而人口统计学因素（年龄、性别）、地理区域和驱虫史未显示统计学显著关联。 讨论： 在少数成功基因分型的分离株中检测到了先前在人类和多种动物中报道的聚合群，但寺庙流浪猫的感染来源、传播方向及其作为储存宿主的地位尚无法确定。

论文解读

研究背景与目的

贾第鞭毛虫病（giardiasis）是一种广泛分布的肠道寄生虫病，具有重要的兽医和公共卫生意义，被公认为最常见的食源性和水源性疾病之一。其病原体十二指肠贾第虫（Giardia duodenalis）是贾第属中唯一已知可感染人类及多种家养和野生哺乳动物的物种。尽管形态相似，分子分型已将该物种划分为八个不同的基因聚合群（assemblages A–H）。其中，聚合群A和B被认为具有人兽共患性，宿主范围广泛；而聚合群C–H则表现出宿主特异性，如聚合群F主要感染猫科动物。在泰国，流浪动物数量庞大，它们常缺乏兽医关注并暴露于广泛的环境污染中，增加了寄生虫感染的易感性。这些流浪猫常栖居于寺庙等公共场所，增加了与人类及其他动物的接触机会。然而，泰国关于猫 G. duodenalis 感染的流行病学信息仍然有限，已有的少数研究大多仅依赖显微镜检查或粪抗原检测，缺乏分子特征分析，而流浪猫群体此前从未被系统调查。因此，本研究旨在确定曼谷寺庙流浪猫中 G. duodenalis 的感染率，利用分子方法鉴定所发现的基因聚合群，并探讨与PCR阳性相关的因素。

研究设计与方法

研究人员于2015年6月至12月在泰国曼谷开展了一项横断面研究。基于分层随机抽样方法，从24个区中随机选择了43座拥有可接触流浪猫群体的寺庙，每区1至3座。根据Yamane公式计算最小样本量，并考虑到潜在的样本损失或降解，目标样本量增加至500份。粪便样本从每只猫的直肠单独采集，采样猫被临时标记以确保每只猫仅贡献一份样本。样本在4°C条件下运输至泰国农业大学兽医学院寄生虫学系。研究人员使用结构化问卷采访僧侣或动物看护者，收集年龄、性别、驱虫史、人类接触和采样区域等信息。人类接触的操作性定义基于猫对人类的日常行为耐受性。

在实验室检测方面，研究人员使用E.Z.N.A.? Stool DNA Kit从约200 mg粪便样本中提取基因组DNA。分子检测采用针对小亚基核糖体RNA（SSU rRNA）基因的巢式PCR进行初步筛查，阳性样本随后通过扩增谷氨酸脱氢酶（gdh）和磷酸丙糖异构酶（tpi）位点进行基因分型。PCR产物经纯化后送AITbiotech公司测序。序列通过BLAST与GenBank参考序列比对，并使用MEGA 12软件基于Tamura 3参数模型构建最大似然系统发育树，进行1,000次自举重复。统计分析使用IBM SPSS Statistics 27版本，采用Wilson评分法估算95%置信区间，单变量分析使用Pearson卡方或Fisher精确检验，P < 0.25的变量进入多变量逻辑回归模型。

研究结果

3.1 流浪猫中 G. duodenalis 的感染率

基于SSU rRNA基因的巢式PCR扩增，所采样流浪猫群体中 G. duodenalis 的总体感染率为10.8%（54/500；95% CI: 8.4–13.8%）。该寄生虫在24个被调查区中的12个（50.0%）和43个被采样寺庙中的19个（44.2%）被检测到。感染率最高的区为邦苏区（Bang Sue），达30.0%（3/10），其次是空三华区（Khlong Sam Wa）的27.6%（8/29）和赛迈区（Sai Mai）的27.3%（9/33）。

3.2 G. duodenalis 的序列分析与基因分型

代表性SSU rRNA序列与参考序列KT595651显示100%一致性。在54份阳性样本中，分别有7.4%（4/54）和11.1%（6/54）在 gdh 和 tpi 位点成功分型。总体而言，共对8个分离株完成了基因分型：2个仅在 gdh 位点鉴定，4个仅在 tpi 位点鉴定，2个获得了多位点基因分型数据。聚合群B（亚聚合群BIV）在50.0%（4/8）的基因分型分离株中被鉴定，聚合群F在37.5%（3/8）中被检测到，聚合群A（亚聚合群AI）在单个分离株（12.5%）中被发现。BLAST序列一致性与系统发育分析结果完全一致。所有获得的序列已提交至GenBank数据库，登录号为PZ715188–PZ715196（SSU rRNA）、PZ587430–PZ587433（gdh）和PZ587434–PZ587439（tpi）。

3.3 与 G. duodenalis PCR阳性相关的因素

在多变量模型中，缺乏人类接触被确定为与较高PCR阳性几率相关的探索性因素（aOR = 8.80；95% CI: 2.15–35.98；P = 0.002）。尽管性别和地理区域通过了单变量筛选，但在最终模型中均未保留为显著预测因子（P = 0.128和P = 0.068）。年龄组和驱虫史与 G. duodenalis PCR阳性无显著关联。

讨论与结论

本研究中流浪猫的 G. duodenalis 感染率（10.8%）与澳大利亚（10.1%）、中国南方（9.8%）和西班牙（9.2%）的发现相当，但低于德国（17.9%）、墨西哥（25.0%）、约旦（27.9%）和孟加拉国（29.9%）的报道。泰国国内的研究也显示出显著差异，从0.3%到27.3%不等，这可能与诊断方法的敏感性差异有关。

在成功基因分型的分离株中，人兽共患聚合群B是最常检测到的聚合群，其次是猫适应性聚合群F和聚合群A。所有成功分型的分离株分别属于亚聚合群AI和BIV，两者均被认为具有较高的人兽共患潜力。这一发现在泰国具有特殊的流行病学意义，因为这两个亚聚合群已在泰国的人类、家养动物和野生动物中被发现。此外，泰国还报道过一例人类感染猫适应性聚合群F的病例。

缺乏人类接触与PCR阳性相关这一发现可能受社会生态、环境和营养因素的影响。避免人类接触的流浪猫可能表现出更野性的行为，依赖 scavenging（ scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， scavenging， 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