Ambrosio, Monica (2023) Development and validation of BIOVITAE® lamps use in the milking parlor to control buffalo mastitis in relation to animal welfare and antibiotic resistance issue. [Tesi di dottorato]

Anteprima

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Abstract

The Mediterranean buffalo (Bubalis bubalis) is the second most important livestock species after the bovine (Bos taurus) (IDF, 2007). This large bovid is widely distributed, especially in Southern Italy, and it is an important economic resource for milk production to produce Mozzarella di Bufala Campana (MBC), a traditional cheese of the Campania Region which is renowned worldwide. Breeding lactating buffaloes can result in the occurrence of mastitis cases. Mastitis is an inflammatory condition of the udder that can be caused by physical and biological hazards, genetic factors, and inadequate environmental and management conditions. In the dairy industry, mastitis is almost always caused by pathogenic and environmental bacteria which, under certain favorable conditions, can enter and multiply in the mammary gland, altering its health and function. The presence of mastitis cases in herds is a critical problem for producers, as the inflammation has consequences on animal health, the quality and quantity of produced milk and, on farmers' income (Halasa et al., 2007). Antibiotic therapy plays an important role in the mastitis treatment. Its success depends on both early detection and proper diagnosis, including identification of the pathogen involved in the inflammation, which will determine the choice of appropriate antibiotic therapy (Erskine et al., 2003). The phenomenon of antimicrobial resistance (AMR), becoming widespread in recent years, is a worrying problem, clinically and economically, and requires innovative strategies for antibiotic treatment. Light-based antimicrobial approaches are becoming a growing translational part of antimicrobial treatments in the current age of resistance. The purpose of this PhD project was to evaluate the antimicrobial action of light device with Biovitae® technology in a milking parlor of the chosen buffalo farm. The experimental plan included an in vitro and in vivo study, in which LED devices, supplied by the Italian company Nextsense, were tested. Through a special combination of frequencies in the visible spectrum (VIS), LED devices create a system of multispectral interfering waves that result in an antimicrobial effect. In Chapter 1, the in vitro use of Master light strip and the light bulb was described. We tested these lights on three bacterial strains previously isolated from the buffalo farm: Escherichia coli (E. coli) and Staphylococcus microti (S. microti) strains, isolated from milk samples, and Staphylococcus aureus (S. aureus) strain isolated from worker’s hand swab. The effect of light exposure resulted in a statistically significant (P ≤0.05) reduction in bacterial growth for E. coli and S. aureus strains after 4 hours of exposure to both the Master light strip and the bulb. The bacterial reduction for the S. microti strain was not statistically significant after 4 hours of exposure to both light devices. Given the link between the excitation of endogenous porphyrins and reactive oxygen species (ROS) production, Dithiothreitol (DTT) and Glutathione (GSH) reagents were tested to regulate intracellular redox reactions and to defend the bacterial cell viability from ROS production. DTT or GSH presence in bacterial cultures showed a protective effect expressed by the reduction of photodynamic microbicidal activity induced by Biovitae® light. In vivo study, reported in Chapter 2, described the monitoring of light effect performing ten samplings in milking parlor. Differences in the bacteria load and number of isolated bacterial genus and species were detected, comparing the results before and after the lights installation. The study included the collection of different samples (milk samples, milking parlor surfaces and workers' hands and nostrils swabs), bacteriological examination and proteomic identification of isolated bacteria. In addition, somatic cell count (SCC) and composition analysis were performed for milk samples to detect intramammary infections, mastitis cases, and milk quality changes. No case of clinical mastitis was detected based on SCC analysis and bacteriological examination. Whereases, 22 out of 200 (11%) milk samples were associated with subclinical mastitis (SCM) cases and 124 out of 200 (62%) milk samples with intramammary infection (IMI). The obtained results from the use of antimicrobial device showed that the light progressively reduced the presence of Gram-negative bacteria, whereas the Gram-positive bacterial load fluctuated during all ten sampling. In addition, colony forming unit (CFU) and the number of bacterial genus and species isolated increased significantly when action of Biovitae® light was stopped, demonstrating the antimicrobial role of light in vivo. Bacterial identification by MALDI-TOF-MS led to the detection in milk samples and milking parlor swabs of a little-known staphylococcus species within non-staphylococcus aureus (NAS) group, identified as Staphylococcus microti (Chapter 3). The identification of 53 S. microti strains was further confirmed by whole-genome 16S rRNA sequencing. Their phenotypic resistance profiles were evaluated by a disk diffusion method, and the tetracycline resistance of strains (100%) was also performed by genotypic characterization. Genotypic analysis, evaluated for the tetM and tetK genes by multiplex PCR, detected the presence of the tetM gene in all isolated S. microti strains. In addition, SCC was performed and the association of this value with the presence of S. microti strains showed that 37.9% of the strains isolated from milk were associated with IMI cases and 18.2% with SCM cases. From all collected samples, the predominant Gram-negative bacteria was E. coli, which is generally found in manure, soil, and the farm environment. It often indicates improperly performed hygienic and management conditions. In Chapter 4, we described the isolation of E. coli strains (n.88) and antimicrobial susceptibility profiles which showed a high level of resistance to penicillin (100%) and clindamycin (98.9%). In addition, the strains showed against tetracycline a percentage of 29.5% resistance and a higher of intermediate susceptibility (65.9%) representative of an emerging resistance, probably related to its frequent use in mastitis cases. The genotypic characterization of the most fGram-negative bacteria's most frequently detected genes med on phenotypically tetracycline resistant E coli. By multiplex PCR, we detected the presence of the tetA, tetB, tetC, tetD e tetG genes, alone or in combination. For the E. coli exhibiting intermediate susceptibility to tetracycline no tested genes showed positivity suggesting the need of further studies. In addition, the association for the milk samples of SCC value with E. coli detection showed that 33.9% of samples were categorized as IMI cases and 27.3% as SCM cases. In parallel with the rise of AMR, innovative approaches are being developed. One possible addition to the current range of treatments is the use of light with antimicrobial activity, able to eliminate a range of common pathogens. The results herein obtained from the antimicrobial photodynamic action produced by the Biovitae® LED devices are encouraging both in vitro and in vivo, when installed in milking parlor. We can conclude that the light alone has the advantages of being easy to install, useful for lighting and lower electricity consumption, and associated with correct routine cleaning practices, could be a valid and innovative approach to controlling bacteria capable of causing mastitis.

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