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Mastitis is one of the most impacting diseases in dairy farming, and its sensitive and specific detection is therefore of the greatest importance. The clinical evaluation of udder and mammary secretions is typically combined with the milk Somatic Cell Count (SCC) and often accompanied by its bacteriological culture to identify the causative microorganism. In a constant search for improvement, several non-enzymatic milk proteins, including milk amyloid A (M-SAA), haptoglobin (HP), cathelicidin (CATH), and lactoferrin (LF), have been investigated as alternative biomarkers of mastitis for their relationship with mammary gland inflammation, and immunoassay techniques have been developed for detection with varying degrees of success. To provide a general overview of their implementation in the different dairy species, we carried out a systematic review of the scientific literature using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines. Our review question falls within the type “Diagnostic test accuracy questions” and aims at answering the diagnostic question: “Which are the diagnostic performances of mastitis protein biomarkers investigated by immunoassays in ruminant milk?”. Based on 13 keywords combined into 42 searches, 523 manuscripts were extracted from three scientific databases. Of these, 33 passed the duplicate removal, title, abstract, and full-text screening for conformity to the review question and document type: 78.8% investigated cows, 12.1% sheep, 9.1% goats, and 6.1% buffaloes (some included more than one dairy species). The most frequently mentioned protein was M-SAA (48.5%), followed by HP (27.3%), CATH (24.2%) and LF (21.2%). However, the large amount of heterogeneity among studies in terms of animal selection criteria (45.5%), index test (87.9%), and standard reference test (27.3%) resulted in a collection of data not amenable to meta-analysis, a common finding illustrating how important it is for case definitions and other criteria to be standardized between studies. Therefore, results are presented according to the SWiM (Synthesis Without Meta-analysis) guidelines. We summarize the main findings reported in the 33 selected articles for the different markers and report their results in form of comparative tables including sample selection criteria, marker values, and diagnostic performances, where available. Finally, we report the study limitations and bias assessment findings.

Staphylococci and streptococci are common causes of intramammary infection in small ruminants, and reliable species identification is crucial for understanding epidemiology and impact on animal health and welfare. We applied MALDI-TOF MS and gap PCR-RFLP to 204 non-aureus staphylococci (NAS) and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis. The top identified NAS was Staphylococcus epidermidis (28.9%) followed by Staph. chromogenes (27.9%), haemolyticus (15.7%), caprae, and simulans (6.4% each), according to both methods (agreement rate, AR, 100%). By MALDI-TOF MS, 13.2% were Staph. microti (2.9%), xylosus (2.0%), equorum, petrasii and warneri (1.5% each), Staph. sciuri (now Mammaliicoccus sciuri, 1.0%), arlettae, capitis, cohnii, lentus (now M. lentus), pseudintermedius, succinus (0.5% each), and 3 isolates (1.5%) were not identified. PCR-RFLP showed 100% AR for Staph. equorum, warneri, arlettae, capitis, and pseudintermedius, 50% for Staph. xylosus, and 0% for the remaining NASM. The top identified streptococcus was Streptococcus uberis (89.5%), followed by Strep. dysgalactiae and parauberis (3.5% each) and by Strep. gallolyticus (1.8%) according to both methods (AR 100%). Only one isolate was identified as a different species by MALDI-TOF MS and PCR-RFLP. In conclusion, MALDI-TOF MS and PCR-RFLP showed a high level of agreement in the identification of the most prevalent NAS and streptococci causing small ruminant mastitis. Therefore, gap PCR-RFLP can represent a good identification alternative when MALDI-TOF MS is not available. Nevertheless, some issues remain for Staph. haemolyticus, minor NAS species including Staph. microti, and species of the novel genus Mammaliicoccus.

Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

The cows receiving antibiotics for intra-mammary infection (IMI) produce milk that cannot be marketed. This is considered waste milk (WM), and a convenient option for farmers is using it as calf food. However, adding to the risk of selecting resistant bacteria, residual antibiotics might interfere with the gut microbiome development and influence gastrointestinal health. We assessed the longitudinal effect of unpasteurized WM containing residual cefalexin on calf intestinal health and fecal microbiota in an 8-week trial. After 3 days of colostrum, six calves received WM and six calves received bulk tank milk (BM) for 2 weeks. For the following 6 weeks, all 12 calves received milk substitute and starter feed. Every week for the first 2 weeks and every 2 weeks for the remaining 6 weeks, we subjected all calves to clinical examination and collected rectal swabs for investigating the fecal microbiota composition. Most WM calves had diarrhea episodes in the first 2 weeks of the trial (5/6 WM and 1/6 BM), and their body weight was significantly lower than that of BM calves. Based on 16S rRNA gene analysis, WM calves had a lower fecal microbiota alpha diversity than that in BM calves, with the lowest p-value at Wk4 (p < 0.02), 2 weeks after exposure to WM. The fecal microbiota beta diversity of the two calf groups was also significantly different at Wk4 (p < 0.05). Numerous significant differences were present in the fecal microbiota taxonomy of WM and BM calves in terms of relative normalized operational taxonomic unit (OTU) levels, affecting five phyla, seven classes, eight orders, 19 families, and 47 genera. At the end of the trial, when 6 weeks had passed since exposure to WM, the phyla Bacteroidetes, Firmicutes, and Saccharibacteria were lower, while Chlamydiae were higher in WM calves. Notably, WM calves showed a decrease in beneficial taxa such as Faecalibacterium, with a concomitant increase in potential pathogens such as Campylobacter, Pseudomonas, and Chlamydophila spp. In conclusion, feeding pre-weaned calves with unpasteurized WM containing antibiotics is related to a higher incidence of neonatal diarrhea and leads to significant changes in the fecal microbiota composition, further discouraging this practice in spite of its short-term economic advantages.

We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled “Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats”. After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243.

We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled "Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats". After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243.

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 milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats. SIGNIFICANCE: The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value.