USDA-ARS Animal Disease Research Unit, Pullman, WA, 99164, USA.
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, 99164, USA.
BMC Vet Res. 2024 Nov 1;20(1):502. doi: 10.1186/s12917-024-04342-y.
Flock-level prevalence and characterization of Mycoplasma ovipneumoniae is determined almost exclusively using nasal swabbing followed by molecular detection with either quantitative PCR or multi-locus sequence typing. However, the diagnostic performance and efficiency of swabbing the nasal passage compared to other anatomical locations has not been determined within sheep populations. The goal of this research was to assess the diagnostic capability of nasopharyngeal swabs in comparison to nasal swabs for the detection of Mycoplasma ovipneumoniae.
Nasal and nasopharyngeal swabs were collected during a controlled exposure study of domestic sheep with Mycoplasma ovipneumoniae. Both swab types were then analyzed via conventional and quantitative PCR. This dataset showed that the use of nasopharyngeal swabs in lieu of nasal swabs resulted in higher sensitivity, reduced inhibition during quantitative PCR, and higher bacterial copy numbers per swab. Moreover, it was demonstrated that diagnostic sensitivity could be further increased during quantitative PCR via ten-fold dilution of the extracted DNA. To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock.
Results demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. This demonstrates that, in addition to anatomical location, the presence of inhibitory components in swab extracts also strongly influences diagnostic performance.
支原体肺炎的群体流行率和特征主要通过鼻腔拭子采集，然后进行分子检测，包括定量 PCR 和多位点序列分型。然而，在绵羊群体中，尚未确定鼻腔拭子与其他解剖部位相比的诊断性能和效率。本研究的目的是评估鼻后滴注拭子与鼻腔拭子在检测支原体肺炎方面的诊断能力。
在一项支原体肺炎绵羊的人工感染控制研究中，采集了鼻腔和鼻后滴注拭子。然后，通过常规 PCR 和定量 PCR 分析这两种拭子类型。该数据集表明，使用鼻后滴注拭子代替鼻腔拭子可提高敏感性，减少定量 PCR 中的抑制作用，以及每个拭子的细菌拷贝数更高。此外，通过对提取的 DNA 进行十倍稀释，证明了定量 PCR 可以进一步提高诊断敏感性。为了在自然感染的动物中证实这些观察结果，我们在一个采用鼻腔和鼻后滴注拭子技术的绵羊生产群体中进行了一项现场研究。使用相同的分子技术评估提取的 DNA，通过 rpoB 或 16S rRNA 基因的测序来确认支原体肺炎的检测。在自然感染的羊群中，鼻后滴注拭子和模板处理方法也观察到了类似的改进。
与鼻腔拭子相比，鼻后滴注拭子采样可提高诊断的敏感性和特异性。因此，在诊断支原体肺炎的存在时，应考虑采用鼻后滴注拭子进行替代现场测试策略。重要的是，采集后样本的处理方式会影响定量 PCR 的敏感性，洗脱 DNA 模板的稀释可使计算出的敏感性提高一倍。这表明，除了解剖位置外，拭子提取物中抑制成分的存在也会强烈影响诊断性能。
