目的:约翰氏病的死前诊断,由鸟分枝杆菌亚种引起。副结核病(MAP),通常是通过粪便培养来实现的,PCR,或血清学测试,但是对于哪些样本对约翰的疾病呈阳性的一致性通常很差,敏感性也很低,特别是在早期感染中。潜在的解决方案:分枝杆菌细胞含有在感染过程中引起抗体的霉菌酸衍生物的非常复杂的特征性混合物;这已用于检测人类的感染。这里,我们探索了其在提供区分感染的动物与接种疫苗的动物(DIVA测定)中的应用。
方法:通过粪便PCR和商业血清ELISA,使用ELISA对MAP阳性的牛血清测量对不同类别的霉菌酸衍生物的抗体反应。或者只是通过PCR,来自没有约翰病史的牛群的动物,牛结核病反应器,接种卡介苗,接种卡介苗和牛分枝杆菌感染,和Gudair接种疫苗的动物。
结果:表现最好的抗原,ZAM295和ST123-后者是MAP细胞中存在的分子,而不是牛分枝杆菌-达到了75%和62.5%的灵敏度,分别,对于粪便PCR和商业MAP血清ELISA阳性的动物血清,特异性为94%,与80个无病史阴性相比。将单独测定的结果与两种抗原(ST123和JRRR121)组合将灵敏度/特异性提高到75/97.5%。在相同的截止日期,接种Gudair或BCG疫苗和bTB反应器的动物表现出相似的特异性。在接种BCG但感染牛分枝杆菌的动物中的特异性下降到85%。结合两种抗原的结果,对全套80份PCR阳性样品的敏感性/特异性为37.5/97.5%,检测到30个阳性,而IDEXX为16个阳性。
结论:使用合成脂质的血清ELISA可有效区分MAP阴性牛样品和PCR和商业MAP血清诊断阳性牛样品,没有Gudair或BCG疫苗的干扰。它鉴定出的PCR阳性几乎是商业血清诊断的两倍,提供早期检测感染的可能性。
OBJECTIVE: Ante-mortem diagnosis of Johne\'s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne\'s disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (
DIVA assay) for Johne\'s disease in cattle.
METHODS: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne\'s disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals.
RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX.
CONCLUSIONS: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.