Alvarez A H, Gutiérrez-Ortega A, Gómez-Entzin V, Pérez-Mayorga G, Naranjo-Bastién J, González-Martínez V, Milián-Suazo F, Martínez-Velázquez M, Herrera-Rodríguez S, Hinojoza-Loza E
Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco A.C., Av. Normalistas 800, C.P. 44270, Guadalajara, Jal., Mexico.
Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco A.C., Av. Normalistas 800, C.P. 44270, Guadalajara, Jal., Mexico.
Microb Pathog. 2017 Jul;108:114-121. doi: 10.1016/j.micpath.2017.05.012. Epub 2017 May 6.
Bovine tuberculosis (bTB) is usually diagnosed in vivo and ex vivo on the basis of delayed hypersensitivity reactions with a complex pool of antigens named bovine tuberculin (PPDB). The IFN-γ release assay (IGRA) for bTB is a blood-based assay that improves detection of infected cattle at early stages that escape skin testing. Improvements to IFN-γ testing with specific proteins have been performed to increase sensitivity. DosR regulon-related antigens are well known mycobacterial proteins expressed during the non-replicative phases of infection, this has been useful to improve the diagnosis of subclinical forms of TB in suspected individuals. Transcripts of DosR genes mb2054c, mb2057c, and mb2660c have been identified by our group in lymph nodes of IFN-γ test negative cattle. This led us to hypothesize that DosR-related proteins may potentiate the IFN-γ response to PPDB in animals with a false negative IFN-γ test, making evident subclinical infection. Three hundred animals were evaluated by means of IGRA and post-mortem microbiological analysis of tissue samples to validate M. bovis infection. We found that 176 out of 300 animals showed an overall increased OD in complemented IGRA with two purified protein cocktails in comparison to PPDB alone, and were scrutinized for a subclinical infection; thirty percent when PPDB was supplemented with a cocktail of four DosR antigens, and 70% when PPDB was supplemented with a cocktail of six antigens (four DosR and two RD1 antigens). Forty five animals showed a substantial IFN-γ overproduction but remained negative, and 40 animals changed the result to a positive test. Only 18 out of 176 IFN-γ high producing animals were also positive to M. bovis isolation. Fifty seven animals with no visible lesions at slaughter and with a negative IGRA test result contained M. bovis DNA in tissue samples. In conclusion, Mb1762c, Mb2054c, Mb2057c, and Mb2660c have the potential to increase sensitivity of the IFN-γ in vitro test for bTB diagnosis when supplemented to PPDB.
牛结核病（bTB）通常基于对一组名为牛结核菌素（PPDB）的复合抗原的迟发型超敏反应，在体内和体外进行诊断。用于bTB的γ-干扰素释放试验（IGRA）是一种基于血液的检测方法，可提高对早期逃避皮肤检测的感染牛的检测能力。已对使用特定蛋白质的γ-干扰素检测进行改进以提高灵敏度。DosR调控子相关抗原是在感染的非复制阶段表达的众所周知的分枝杆菌蛋白，这有助于改善疑似个体中亚临床型结核病的诊断。我们的研究小组在γ-干扰素检测呈阴性的牛的淋巴结中鉴定出了DosR基因mb2054c、mb2057c和mb2660c的转录本。这使我们推测，DosR相关蛋白可能会增强γ-干扰素对PPDB的反应，从而在γ-干扰素检测呈假阴性的动物中使亚临床感染显现出来。通过IGRA和对组织样本进行死后微生物学分析对300只动物进行评估，以验证牛分枝杆菌感染。我们发现，300只动物中有176只在使用两种纯化蛋白混合物补充的IGRA中，与单独使用PPDB相比，总体光密度增加，并对其进行亚临床感染检查；当PPDB添加四种DosR抗原混合物时为30%，当PPDB添加六种抗原（四种DosR和两种RD1抗原）混合物时为70%。45只动物显示γ-干扰素大量产生但仍为阴性，40只动物的检测结果变为阳性。在176只高产生γ-干扰素的动物中，只有18只对牛分枝杆菌分离也呈阳性。57只在屠宰时无可见病变且IGRA检测结果为阴性的动物，其组织样本中含有牛分枝杆菌DNA。总之，Mb1762c、Mb2054c、Mb2057c和Mb已鉴定出DosR基因mb2054c、mb2057c和mb2660c的转录本。这使我们推测，DosR相关蛋白可能会增强γ-干扰素对PPDB的反应，从而在γ-干扰素检测呈假阴性的动物中使亚临床感染显现出来。通过IGRA和对组织样本进行死后微生物学分析对300只动物进行评估，以验证牛分枝杆菌感染。我们发现，300只动物中有176只在使用两种纯化蛋白混合物补充的IGRA中，与单独使用PPDB相比，总体光密度增加，并对其进行亚临床感染检查；当PPDB添加四种DosR抗原混合物时为30%，当PPDB添加六种抗原（四种DosR和两种RD1抗原）混合物时为70%。45只动物显示γ-干扰素大量产生但仍为阴性，40只动物的检测结果变为阳性。在176只高产生γ-干扰素的动物中，只有18只对牛分枝杆菌分离也呈阳性。57只在屠宰时无可见病变且IGRA检测结果为阴性的动物，其组织样本中含有牛分枝杆菌DNA。总之，Mb1762c、Mb2054c、Mb2057c和Mb2660c在补充到PPDB中时，有可能提高用于bTB诊断的γ-干扰素体外检测的灵敏度。 2660c在补充到PPDB中时，有可能提高用于bTB诊断的γ-干扰素体外检测的灵敏度。
