Evaluation of milk cathelicidin for detection of dairy sheep mastitis

Abstract

Mastitis due to intramammary infections is one of the most detrimental diseases in dairy sheep farming, representing a major cause of reduced milk productions and quality losses. In particular, subclinical mastitis presents significant detection and control problems, and the availability of tools enabling its timely, sensitive, and specific detection is therefore crucial. We have previously demonstrated that cathelicidins, small proteins implicated in the innate immune defense of the host, are specifically released in milk of mastitic animals by both epithelial cells and neutrophils. Here, we describe the development of an ELISA for milk cathelicidin and assess its value against somatic cell counts (SCC) and bacteriological culture for detection of ewe mastitis. Evaluation of the cathelicidin ELISA was carried out on 705 half-udder milk samples from 3 sheep flocks enrolled in a project for improvement of mammary health. Cathelicidin was detected in 35.3% of milk samples (249/705), and its amount increased with rising SCC values. The cathelicidin-negative (n=456) and cathelicidin-positive (n=249) sample groups showed a clear separation in relation to SCC, with median values of 149,500 and 3,300,000 cells/mL, respectively. Upon bacteriological culture, 20.6% (145/705) of the milk samples showed microbial growth, with coagulase-negative staphylococci being by far the most frequent finding. A significant proportion of all bacteriologically positive milk samples were positive for cathelicidin (110/145, 75.9%). Given the lack of a reliable gold standard for defining the true disease status, sensitivity (Se) and specificity (Sp) of the cathelicidin ELISA were assessed by latent class analysis against 2 SCC thresholds and against bacteriological culture results. At an SCC threshold of 500,000 cells/mL, Se and Sp were 92.3 and 92.3% for cathelicidin ELISA, 89.0 and 94.9% for SCC, and 39.4 and 93.6% for bacteriological culture, respectively. At an SCC threshold of 1,000,000 cells/mL, Se and Sp were 93.3 and 91.9% for cathelicidin ELISA, 80.0 and 97.1% for SCC, and 39.4 and 93.5% for bacteriology, respectively. In view of the results obtained in this study, the measurement of cathelicidin in milk by ELISA can provide added Se while maintaining a high Sp and may therefore improve detection of subclinical mastitis.

Relationship of Late Lactation Milk Somatic Cell Count and Cathelicidin with Intramammary Infection in Small Ruminants

“…Looking at ewes, in our previous study the test performances of CATH in mid-lactation animals were already slightly better than SCC [28]. This study demonstrates that the diagnostic advantage of CATH over SCC increases even further in late lactation ewes.…”

“…When considering that BC on a single milk sample has a theoretical sensitivity of 42.90% in conditions comparable to the ones applied in this study [17], it is plausible that the 25.08% of sheep milk samples positive to CATH and with SCC above 900 × 10 3 cells/mL (the first three quartiles, i.e., 75% of the distribution) were actually affected by IMI. Accordingly, the test performances of CATH in sheep might even be underestimated when considering the limited sensitivity of the gold standard BC [7,17,18,28]. In goats, on the other hand, IMI prevalence was 15.25% according to BC, but 47.98% of samples were CATH-positive, with median SCC values around 1000 × 10 3 cells/mL.…”

“…With the aim of improving the sensitivity and specificity of mastitis detection, we developed a CATH ELISA, building on the sheep gene sequences. The assay showed good diagnostic performances in comparison to SCC and BC in mid-lactation ewes and cows [28,29]. The CATH assay was also demonstrated to be suitable for the detection of cathelicidins in goat milk when physiological levels were assessed over a full lactation year in comparison to the SCC [12].…”

Detection of Cathelicidin-1 in the Milk as an Early Indicator of Mastitis in Ewes

“…Addis et al [8] described the development of an ELISA test to detect cathelicidins in milk. With further development, a rapid test may be produced for animal-side use.…”

“…Among those, detection of cathelicidin has significant advantages as a mammary gland inflammation biomarker for diagnosing the infection. Previous works have already pointed out the value of cathelicidins for mastitis detection in ewes [7,8].…”