Abstract
BackgroundIn the postpartum cow, early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods. Cervico-vaginal mucus (CVM) is an easy to collect potentially informative source of biomarkers for the diagnosis and prognosis of uterine disease in cows. Here, we report an improved method for processing CVM from postpartum dairy cows for the measurement of immune biomarkers. CVM samples were collected from the vagina using gloved hand during the first two weeks postpartum and processed with buffer alone or buffer containing different concentrations of the reducing agents recommended in standard protocols: Dithiothriotol (DTT) or N-Acetyl-L-Cysteine (NAC). Total protein was measured using the bicinchoninic acid (BCA) assay; interleukin 6 (IL-6), IL-8 and α1-acid glycoprotein (AGP) were measured by ELISA.ResultsWe found that use of reducing agents to liquefy CVM affects protein yield and the accuracy of biomarker detection. Our improved protocol results in lower protein yields but improved detection of cytokines and chemokines. Using our modified method to measure AGP in CVM we found raised levels of AGP at seven days postpartum in CVM from cows that went on to develop endometritis.ConclusionWe conclude that processing CVM without reducing agents improves detection of biomarkers that reflect uterine health in cattle. We propose that measurement of AGP in CVM during the first week postpartum may identify cows at risk of developing clinical endometritis.
Highlights
In the postpartum cow, early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods
Measurement IL6, IL8 and acid glycoprotein (AGP) Our modified protocol resulted in the detection of significantly higher levels of both interleukin 6 (IL-6) and IL-8 in postpartum Cervico-vaginal mucus (CVM) per ml of mucus (P < 0.001) (Fig. 1b and c)
Accounting for total protein concentration in each sample, the concentration of IL-6 and IL-8 per mg of total protein was significantly higher in the modified protocol compared to the standard protocol (P < 0.001) (Fig. 1d and e)
Summary
Early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods. E.g. interleukin 6 (IL-6), chemokines e.g. IL-8 and acute phase proteins e.g. α1-Acid Glycoprotein (AGP), produced by endometrial epithelial cells and local immune populations are increased in inflamed uterine tissue and in vitro models [6,7,8]. These inflammatory biomarkers have been detected in uterine mucus and CVM [8,9,10]. The overall objective of the Adnane et al BMC Veterinary Research (2018) 14:297 current study is to improve processing of postpartum CVM for measuring candidate biomarkers that may predict uterine inflammation and disease
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