UNDERSTANDING AND DETECTING DAIRY RUMINANT MASTITIS: RECENT FINDINGS ON MICROBIAL IDENTIFICATION METHODS, DIAGNOSTIC MARKERS, AND THE MICROBIOME

Mastitis is one of the most economically impactful diseases in dairy farming. It may cause severe qualitative-quantitative losses in milk production, adversely affect animal welfare, and lead to early culling of animals. Mastitis can present itself in different forms, often due to the different causative agents. Two main manifestations of the disease are clinical, which is easier to detect due to the use of semi-quantitative techniques or simple examination of the udder, and subclinical , which has no obvious symptoms and requires milk testing to discover mastitis indicators. The focus of this thesis work is to understand dairy ruminant mastitis with a focus on recent findings on microbial identification methods, diagnostic markers of mastitis, and the impact of intramammary antibiotic treatment due to the presence of mastitis on the gut microbiome. The first study was focused on improving the identification of etiologic agents, focusing on the highly prevalent non-aureus staphylococci (NAS), with the application of PCR–RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) and MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) for improving the detection of intramammary infections (IMI). We applied MALDI‐TOF MS and gap PCR–RFLP to 204 NAS and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis to compare their respective results. The study revealed a high level of agreement between MALDI‐TOF MS and PCR–RFLP in identifying the most prevalent NASM and streptococci causing small ruminant mastitis. The second study aimed to conduct a systematic review to synthesize all the literature on the topic and improve the understanding of the diagnostic accuracy of protein marker-based immunoassays for mastitis detection. Currently, the most widely used tool for monitoring mastitis is the somatic cell count (SCC), expressed as the number of cells per ml of milk. Using SCC as a monitor has two major drawbacks. First, it has low specificity, and this limitation is particularly relevant in small ruminants, especially goats, as the SCC increases physiologically with parity and lactation number. The other major drawback is the difficulty of using this system to monitor mastitis in dairy herds of non-bovine species, for which clear reference values are unavailable. This has led to the search for new markers with higher specificity and suitability for easy measurements. Among the most studied are the protein molecules produced by the infected host in the context of inflammation and released in the milk, which makes them directly and easily detectable by techniques that exploit antigen-antibody interaction (immunoassay). To provide a general overview of their application 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. Based on 13 keywords combined into 42 searches, we extracted 523 manuscripts from three scientific databases. After screening for duplicates and pertinence, we summarized the main findings in 33 selected articles for the different markers. Where available, we reported their results in comparative tables, including sample selection criteria, marker values, and diagnostic performances. Finally, we reported the study limitations and bias assessment findings. The third study addressed the impact of mastitis on the calf gut microbiome, particularly concerning the impact of calf feeding with waste milk (WM). The risks associated with using WM as a feed source for calves have recently been highlighted. WM is a by-product of the dairy sector that, due to its physical, chemical, and microbiological properties and the presence of antibiotic residues from the treatment of lactating cows, cannot be marketed for human consumption. This milk may be a resource for calf feeding. Although WM represents an economically advantageous alternative and offers a viable solution to the environmental disposal of this product, it may present risks in terms of antibiotic resistance selection and may impair the healthy development of the calf gut microbiota. In particular, the administration of WM containing antimicrobial residues could contribute to select and maintain resistant bacteria in the animal's gut. In addition to presenting issues in terms of health and management of the animals themselves, this could pose a public health risk: calves, by becoming reservoirs of resistant bacteria, would contribute to the spread of these microorganisms through the environmental release of their feces. The trial assessed the longitudinal effect of unpasteurized WM containing residual cefalexin monohydrate on calf intestinal health and fecal microbiota in an 8-week trial. The feeding trial included 12 male Holstein Friesian calves. After colostrum, which lasted 3 days, 6 calves were fed waste milk (WM group), and 6 calves were fed normal bulk tank milk (NM group) for two weeks. At the end of this first phase, all calves were fed reconstituted whey powder and starter feed, according to standard farm protocols, for another six weeks. For the first two weeks, and then every two weeks until the end of the trial, calves were monitored and weighed, and fecal and swab samples were taken. Our results suggest that feeding pre-weaned calves with unpasteurized WM containing antibiotics leads to significant changes in the fecal microbiota composition, further discouraging this practice despite its short-term economic advantages.