Aminzadeh Mohammad Javad, Hashemi Khadijeh, Rahmani Hamideh Kalateh, Khaleghnia Narges, Azizzadeh Mohammad, Mirshokraei Pezhman
Department of Clinical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.
BMC Vet Res. 2025 Apr 9;21(1):256. doi: 10.1186/s12917-025-04660-9.
Brucellosis is a zoonotic disease occurring worldwide. Brucella melitensis is the main cause of malta fever in humans and a major cause of abortion in sheep. In herd control and prevention programs, applying a suitable noninvasive method for accurate, rapid, and cost-effective monitoring of infected animals is a major concern. This study aimed to investigate lots of pooled sheep milk using qPCR to detect Brucella spp. infection in sheep. By calculating the limit of detection of Brucella in qPCR and microbial culture, the maximum number of pooled milk lots that retained the ability to be identified positively for Brucella was determined. A total of 144 milk samples were collected from the different seropositive sheep herds. The samples were randomly divided into six groups, each further divided into two subgroups, respectively. Then, DNA extraction was performed on 186 pooled and individual samples, followed by qPCR.
The minimum detectable limits for qPCR and microbial culture per ml of milk were 100 and 300 CFU, respectively. Only 40% of the samples in microbial culture tested positive when the concentration decreased to 200 CFU. The results of qPCR indicated that four pools of 24 tested positive, whereas two pools tested negative. After examining the subgroups and individual samples within the two negative groups, it was revealed that all qPCR tests for these samples were negative. In the positive pools, at least one of the samples in the subgroups and corresponding individual samples tested positive. The two positive pools of 24, contained only one individual positive sample in each, indicating that the qPCR test could detect a positive Brucella sample in a pool of 24.
This noninvasive (milk instead of blood), rapid, and cost-effective method can be used to monitor suspected herds to identify infected animals with fewer tests.
布鲁氏菌病是一种在全球范围内发生的人畜共患病。羊种布鲁氏菌是人类马耳他热的主要病因，也是绵羊流产的主要原因。在畜群控制和预防计划中，采用合适的非侵入性方法对感染动物进行准确、快速且经济高效的监测是一个主要关注点。本研究旨在使用qPCR检测绵羊奶样来调查大量混合绵羊奶，以检测绵羊中的布鲁氏菌属感染。通过计算qPCR和微生物培养中布鲁氏菌的检测限，确定了仍能被阳性鉴定出布鲁氏菌的混合奶样的最大数量。总共从不同血清学阳性的绵羊群中收集了144份奶样。这些样本被随机分为六组，每组又进一步分为两个亚组。然后，对186份混合和单个样本进行DNA提取，随后进行qPCR。
每毫升牛奶中qPCR和微生物培养的最低检测限分别为100 CFU和300 CFU。当浓度降至200 CFU时，微生物培养中只有40%的样本检测呈阳性。qPCR结果表明，24份混合样本中有4份检测呈阳性，而2份混合样本检测呈阴性。在检查两个阴性组内的亚组和单个样本后发现，这些样本的所有qPCR检测均为阴性。在阳性混合样本中，亚组中的至少一个样本和相应的单个样本检测呈阳性。24份的两个阳性混合样本中，每个仅包含一个单个阳性样本，这表明qPCR检测可以在24份混合样本中检测到一份阳性布鲁氏菌样本。
这种非侵入性（用奶而非血）、快速且经济高效的方法可用于监测疑似畜群，以通过较少的检测来识别感染动物。
