Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle

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.

Qualitative and quantitative differences in endometrial inflammatory gene expression precede the development of bovine uterine disease

“…The putative gene expression biomarkers can now be validated at the protein level in in samples from cows on additional farms. These findings complement our on-going efforts to identify biomarkers of uterine disease in blood 48 or in vaginal mucus 49 , 50 which will ultimately improve both disease diagnosis and therapeutic intervention.…”

The addition of Lactobacillus spp. negatively affects Mycoplasma bovis viability in bovine cervical mucus

“…The samples were obtained by transrectal massaging of the cranial cervix and placed in 100 ml sterile plastic bottles. The samples were immediately placed on ice in an isothermal box and transported to the laboratory within 1 h [34].…”

A simple and reliable refractometric method to determine the total solids concentration of the cervico-vaginal bovine mucus samples

“…Due to the physical properties of mucus, processing of CVM with reducing agents is routinely recommended before the analysis of solublephase biomarkers [3]. N-acetyl-L-cysteine (NAC) and Dithiothreitol (DTT) are commonly used to homogenize mucus by reducing the disulfide bonds of mucins [3]. However, many immune biomarkers also have disulfide bonds and their detection is likely to be compromised by use of reducing agents [3].…”

“…N-acetyl-L-cysteine (NAC) and Dithiothreitol (DTT) are commonly used to homogenize mucus by reducing the disulfide bonds of mucins [3]. However, many immune biomarkers also have disulfide bonds and their detection is likely to be compromised by use of reducing agents [3]. Alternatively, the non-ionic detergent Triton X-100 is one of the most routinely used agents to solubilise biomembranes [15].…”

“…The postpartum bovine uterus is susceptible to bacterial contamination, viral infection as well as endometritis causing uterine disease [1,3]. Cervico-vaginal mucus (CVM) can be easily collected and does not require special training therefore provides a more accessible resource to assess disease status.…”