Background

Bovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle.

Methods

We developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears.

Results

Four out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAV_TAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection.

Conclusion

PNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.

中文翻译：

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肽核酸荧光原位杂交快速检测牛细胞涂片和组织切片中的牛分枝杆菌

背景

牛结核病是全世界牛和其他物种死亡的主要原因。快速、准确地鉴定分枝杆菌对于控制牛结核病的发生至关重要。

方法

我们开发了一种荧光肽核酸荧光原位杂交（PNA-FISH）方法来检测疑似患有结核病的牛的细胞学涂片和组织切片中的牛分枝杆菌和鸟分枝杆菌。对肺和淋巴结组织涂片进行 PNA-FISH。标准牛分枝杆菌培养物用于标准化探针，牛分枝杆菌使用 50% 甲酰胺，鸟分枝杆菌使用 30%甲酰胺。牛分枝杆菌探针 (MTBCcy3) 在（55 °C 和 40% 甲酰胺）浓度的杂交条件下标准化，在所有细胞学涂片中均呈阳性。

结果

25 个样本中有 4 个在组织切片中检测呈阳性，用 cy3 滤光片（MTBC 探针）观察到亮红色荧光。使用 (MAV _{TAMRA )}鸟分枝杆菌探针未观察到结果，该探针在（55 °C 和 30% 甲酰胺）杂交条件下标准化。在对照组织切片中未观察到荧光。此外，还将结果与其他常用检测方法（例如针对esxA基因的免疫组织化学和聚合酶链反应 (PCR)）的结果进行了比较。所有样本均未检测出鸟分枝杆菌感染呈阳性。

结论

PNA-FISH 可用于获得细胞学印迹涂片和组织切片。与 PCR 相比，它在尸检病例中诊断牛结核病所需的时间更少。