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Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats

ABSTRACT: This investigation reports a differential proteomic analysis of the goat milk to evaluate and understand the protein changes induced by Staphylococcus spp. during a subclinical intramammary infection (IMI). A number of 9 milk samples from multiparous goats were selected for the study, 3 of which in mid-lactation (30-60 Days in milk - DIM) with very low somatic cell count (SCC) from half-udders producing a sterile milk bacterial culture (MLU), 3 of which in late lactation samples (> 250 DIM) with SCC > 2,000,000 cells/mL from half-udders producing a sterile milk bacterial culture for the whole lactation (LHU) and in conclusion 3 in late lactation (> 250 DIM) with SCC > 2,000,000 cells/mL from goat half-udders with a milk bacterial culture repeatedly positive for Staphylococcus aureus (LHS). Samples were analyzed using a shotgun proteomics approach, based on filter-aided sample preparation (FASP) followed by LC-MS/MS and on differential analysis conducted by spectral count approach (1). (1) Pisanu S, Cacciotto C, Pagnozzi D, Puggioni GMG, Uzzau S, Ciaramella P, Guccione J, Penati M, Pollera C, Moroni P, Bronzo V, Addis MF. Proteomic changes in the milk of water buffaloes (Bubalus bubalis) with subclinical mastitis due to intramammary infection by Staphylococcus aureus and by non-aureus staphylococci. Sci Rep, 2019, 9 (1), 15850. Here, we reported both a characterization of goat milk proteins and a panel of differential proteins specific of S. Aureus-infected milk that mostly affected proteins involved in defense response processes and cytoskeleton organization. In conclusion, our results provide a depth characterization of milk proteins in goat samples uninfected and infected with S. Aureus, describe the changes induced by LHU and LHS subclinical intramammary infection and suggest indications to reveal subclinical staphylococcal mastitis in goat by a proteomic investigation of milk.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Capra Hircus (goat)

TISSUE(S): Milk

DISEASE(S): Mastitis

SUBMITTER: Salvatore Pisanu

LAB HEAD: Daniela Pagnozzi

PROVIDER: PXD017243 | Pride | 2020-04-09

REPOSITORIES: Pride

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Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: New perspectives for monitoring and understanding mastitis in dairy goats.

Pisanu Salvatore S Cacciotto Carla C Pagnozzi Daniela D Uzzau Sergio S Pollera Claudia C Penati Martina M Bronzo Valerio V Addis Maria Filippa MF

Journal of proteomics 20200408

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation ...[more]

PMID: 32275959

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Project description:Bovine mastitis, the infection of the mammary gland which leads to great health and economic challenges for dairy farmers is accompanied by dramatic changes in the milk proteome. In this study of naturally occurring mastitis not only have the changes in the milk proteome been quantified in subclinical and clinical mastitis but simultaneous changes in the serum proteome have also been characterised and quantified. Milk and serum samples from healthy dairy cows (n=12) were compared to those of cows with subclinical (n=10) and clinical mastitis (n=112) using TMT label-based proteomic approach. The study included the milk and serum samples taken from thirty-two dairy cows ( kept on private farms located in Croatia. All cows were checked by physical examination. Somatic cells count (SCC) and mastitis test in milk samples were performed. According to the results, cows were assigned into three groups: Group I (control, n=10) consisted of healthy cows with SCC below 400,000 cells/ml on the monthly check-up and a negative mastitis test and without any clinical sign of mastitis. Group II (subclinical mastitis, n=12) comprised cows without clinical signs of mastitis but with SCC above 400,000 cells/ml on the monthly basis and a positive mastitis test at the time of sampling. Group III (clinical mastitis, n=10) consisted of cows with clinical signs of mastitis which include changes in milk appearance (flakes and clots in milk), different stages of udder inflammation (hyperemia, edema, pain, udder enlargement and elevated udder temperature) and disturbance of general health (depression, relaxed cold ears, dehydration, elevated body temperature, increased heart and respiratory rate, decreased ruminal contraction and decreased appetite). Blood samples were taken from v. coccygea and centrifuged at 3000 g for 15 min after clotting for two hours at room temperature. Serum samples were stored at -80°C until analysis. Milk samples were taken aseptically before the morning milking. First few streams were discarded. Teat ends were disinfected with cotton swabs soaked with 70% ethanol. Samples were taken into sterile tubes and transported to laboratory on ice within a few hours.

Project description:Four-times daily milking during the first 3 weeks of lactation elicits an increase in milk yield, which persists through late lactation even after twice-daily milking is imposed. We hypothesized that this milk yield response would be associated with changes in mammary proliferation, apoptosis, and gene expression, which would persist throughout lactation. Six multiparous cows were assigned to unilateral frequent milking (UFM; twice daily milking of the left udder half (2X), four-times daily milking of the right udder half (4X)) on days 1 to 21 of lactation, followed by 2X thereafter. Udder halves initially milked 4X produced more milk than those milked 2X during, and after UFM treatment, through 180 days in milk (DIM). To determine the mechanisms involved in the persistent milk yield response, we obtained mammary biopsies from both udder halves at 21, 23, and 40 DIM. Rates of [3H]-thymidine incorporation into DNA in vitro and mammary cell apoptosis were not affected by UFM or DIM. Using Affymetrix GeneChipM-BM-. Bovine Genome Arrays, we determined that the differential expression (4X vs. 2X) of 18 genes was significantly affected by DIM. Within the group of 18 differentially expressed genes, we identified a cluster of 15 genes with a similar temporal pattern of differential expression. Nine of the genes in the cluster remained differentially expressed at 40 DIM, indicating that they may be involved in the persistent milk yield response. Among the genes in the cluster were chitinase 3-like (CHI3L)-1, clusterin, early growth response (EGR)-1, and sex determining region Y-box (SOX)-4. These genes have been associated with mammary development, differentiation and remodeling; all of which may be functionally related to the increase in milk yield. We conclude that frequent milking during early lactation does not alter mammary growth but is associated with changes in mammary expression of 18 genes. Future experiments will determine the function of these genes in the mammary gland, and will clarify their role in the autocrine regulation of milk production and long-term alteration of mammary function. 36 samples from 6 cows; 3 timepoints

Dataset Information

Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats

Publications

Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: New perspectives for monitoring and understanding mastitis in dairy goats.

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