Almeida Patricia E, Weber Patty S D, Burton Jeanne L, Tempelman Robert J, Steibel Juan P, Zanella Adroaldo J
Immunogenetics Laboratory, Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA.
Vet Immunol Immunopathol. 2007 Dec 15;120(3-4):234-45. doi: 10.1016/j.vetimm.2007.06.028. Epub 2007 Jun 28.
Lameness is a major health issue and likely the single most common cause of pain and discomfort in dairy cattle. Appropriate treatment is delayed or neglected due, in part, to lack of reliable detection. Assessment of cows with lameness is currently limited to subjective visual scoring systems based on locomotion and posture abnormalities. These systems are unreliable to detect lameness, and therefore, a large number of cows remain undiagnosed. The objective of this research was to search for potential biomarkers for lameness-associated painful inflammatory foot lesions in dairy cattle using microarray-based gene expression profiling of peripheral blood mononuclear cells (PBMC). BOTL5 microarrays spotted in duplicate with cDNA representing bovine immune response genes were interrogated with cDNA samples in an eight-array, balanced complete block design with dye swap. Samples from eight lame cows with inflammatory foot lesions and from eight sound cows were pair-matched by age, weight, days in lactation, and pregnancy status at time of PBMC collection and directly compared with each other on individual arrays. Statistical analysis of resulting fluorescence intensity data revealed 31 genes that were putatively differentially expressed in lame versus sound cows (P<0.05). Of these, BLASTn analysis and gene ontology information showed that 28 genes had high similarity or homology to known human and/or rodent genes. Validation of 15 of these genes known to be important in inflammation and pain was carried out using relative quantitative real-time RT-PCR, which confirmed the up-regulation of interleukin (IL)-2 (12.68+/-1.47-fold increase) and IL-10 (2.39+/-0.55-fold increase), matrix metalloproteinase-13 (MMP-13) (10.44+/-1.14-fold increase), and chemokine C-C motif receptor-5 (CCR5) (5.26+/-1.05-fold increase), in lame relative to sound cows (P< or =0.05). Similarly, granulocyte-macrophage colony-stimulating factor receptor alpha chain precursor (GM-CSF-R-alpha) (2.30+/-0.63-fold increase) and IL-4 (2.06+/-0.59-fold increase) showed a tendency (P=0.10) for up-regulation in lame compared to sound cows. PBMC co-expression of IL-2, MMP-13, CCR5 and IL-10, and potentially IL-4 and GM-CSF-R-alpha appears to be a promising, objective sign of lameness-related inflammatory foot lesions in dairy cattle. In conclusion, this study revealed potential biomarkers of the presence of foot lesions that could boost diagnostic accuracy of lameness and, ultimately, help identify animals in need of pain relief.
跛行是一个主要的健康问题，可能是奶牛疼痛和不适的最常见单一原因。部分由于缺乏可靠的检测方法，适当的治疗被延迟或忽视。目前对跛行奶牛的评估仅限于基于运动和姿势异常的主观视觉评分系统。这些系统在检测跛行方面不可靠，因此大量奶牛仍未被诊断出来。本研究的目的是使用外周血单核细胞（PBMC）的基于微阵列的基因表达谱，寻找奶牛跛行相关疼痛性炎症性足部病变的潜在生物标志物。用代表牛免疫反应基因的cDNA一式两份点样的BOTL5微阵列，在八阵列平衡完全区组设计中与cDNA样品进行杂交，并进行染料交换。从八头患有炎症性足部病变的跛行奶牛和八头健康奶牛采集样本，在采集PBMC时按年龄、体重、泌乳天数和妊娠状态进行配对，并在单个阵列上直接相互比较。对所得荧光强度数据的统计分析显示，有31个基因在跛行奶牛和健康奶牛中可能存在差异表达（P<0.05）。其中，BLASTn分析和基因本体信息表明，28个基因与已知的人类和/或啮齿动物基因具有高度相似性或同源性。使用相对定量实时RT-PCR对其中15个已知在炎症和疼痛中起重要作用的基因进行验证，结果证实与健康奶牛相比，跛行奶牛中白细胞介素（IL）-2（增加12.68±1.47倍）、IL-10（增加2.39±0.55倍）、基质金属蛋白酶-13（MMP-13）（增加10.44±1.14倍）和趋化因子C-C基序受体-5（CCR5）（增加5.26±1.05倍）上调（P≤0.05）。同样，与健康奶牛相比，粒细胞-巨噬细胞集落刺激因子受体α链前体（GM-CSF-R-α）（增加2.30±0.63倍）和IL-4（增加2.06±0.59倍）有上调趋势（P=0.10）。PBMC中IL-2、MMP-13、CCR5和IL-10以及可能的IL-4和GM-CSF-R-α的共表达似乎是奶牛跛行相关炎症性足部病变的一个有前景的客观指标。总之，本研究揭示了足部病变存在的潜在生物标志物，这可以提高跛行的诊断准确性，并最终帮助识别需要缓解疼痛的动物。
