Detection of Cathelicidin-1 and Cathelicidin-2 Biomolecules in the Milk of Goats and Their Use as Biomarkers for the Diagnosis of Mastitis

Abstract

The objectives of the present work were as follows: (i) the detection of cathelicidin biomolecules in the milk of individual goats during the early stages of mastitis and their potential use for the diagnosis of mastitis at its early stage and (ii) the evaluation of the presence of cathelicidin proteins in the bulk-tank milk from goat and sheep farms. In an experimental study, after inoculation of Staphylococcus simulans into a mammary gland of goats, bacteriological and cytological examinations of milk samples, as well as proteomics examinations [two-dimensional gel electrophoresis analysis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis] were performed sequentially, from 4 to 48 h post-challenge. Cathelicidin-1 and cathelicidin-2 were detected consistently in milk samples obtained throughout the study, and spot optical densities obtained from PDQuest v.8.0 were recorded. Associations were calculated between the presence of mastitis in a mammary gland at a given timepoint and the detection of cathelicidin proteins in the respective milk sample. All inoculated mammary glands developed mastitis, confirmed by the consistent bacterial isolation from milk samples and the increased somatic cell content therein. Spot optical density of cathelicidin proteins was higher than in samples from contralateral mammary glands. There was a significant association between the presence of mastitis in a mammary gland and the detection of cathelicidin biomolecules in the respective milk sample; the overall accuracy was 81.8% (95% confidence interval: 70.4-90.2%). In a field investigation, the presence of cathelicidin proteins was evaluated in the bulk-tank milk of 32 dairy goat and 57 dairy sheep farms. In this part of the work, no cathelicidin proteins were detected in any bulk-tank milk sample of goat, 0.0% (95% confidence interval: 0.0-10.7%), or sheep, 0.0% (95% confidence interval: 0.0-6.3%), farms.

Keywords: biomarker; cathelicidin-1; cathelicidin-2; diagnosis; goat; mastitis; proteomics; sheep; somatic cell counts.

Figure 1

Summary of the work process in the experimental study (figure produced in BioRender, under academic publication licence no. EN28LJSRU7 (2025)).

Figure 2

Violin plots for spot optical densities of cathelicidin-1 (blue plot) or cathelicidin-2 (red plot) on 2-DE gels obtained from sequential milk whey samples from mammary glands inoculated with S. simulans for 48 h subsequently to challenge; violin plots depict the probability density of the data at different values and compare probability distributions of the optical densities of the two cathelicidin proteins.

Figure 3

Trendlines for spot optical densities of cathelicidin-1 (blue dashed line) or cathelicidin-2 (red dashed line) on 2-DE gels obtained from sequential milk whey samples from mammary glands inoculated with S. simulans and comparison with trendline of somatic cell counts (black dotted line) in the same samples; trendlines for the spot optical densities of the two cathelicidin proteins indicated an early increase in the values, which reached maximum figures after 12 to 24 h post-challenge, in contrast with somatic cell counts, which increased at a later stage and continued to rise as the infection developed further.

Figure 4

Radar-type plot of the time post-challenge, when cathelicidins (coloured dashed line) were first seen on 2-DE gels obtained from sequential milk whey samples from each of the three mammary glands inoculated with S. simulans and comparison with the time post-challenge when increased somatic cell counts (>1.500 × 10⁶ cells mL⁻¹) or increased CMT scores (≥‘2’) (black dotted line) were first recorded in milk samples from the same glands (orange markers indicate the three mammary glands challenged in the study); the plot clearly indicates the early rise in cathelicidin proteins in the milk of inoculated animals (4 h post-challenge), in contrast to the substantially later development of the leucocyte response of the animals (≥16 h post-challenge) (grey lines are gridlines).