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Clostridium species are known for their impact on animal and human health, but also for the spoilage of foodstuffs. Their spores contaminate milk and result in germination and gas production, the latter being particularly evident in the cheeses that suffer severe depreciation. To address this issue, the Primary Production Department of the IZSLER institute in Brescia, Italy conducts the Most Probable Number (MPN) method on bovine milk samples collected from Northern Italian dairies between 2004 and 2023. This approach leverages two semi-quantitative protocols, S2 and S3, to detect Clostridium species spore forms upon customer request. Here, we would like to present an a-posteriori analysis on the results of the S2 and S3 protocols. The goal of this study is to highlight the differences between these two methods and provide evidence of the actual decrease in Clostridium species in raw cow milk over a 20-year period. Our analysis shows that client demand for S2 has progressively decreased, while S3's has remained constant, and both protocols reveal a significant reduction in positives; furthermore, S3's greater sensitivity made it more responsive to environmental changes. This highlights the necessity of choosing the appropriate testing protocol that accounts for both regulatory standards and environmental factors. Overall, our findings underscore the importance of continued monitoring to manage Clostridium species contamination and ensure milk quality. [ABSTRACT FROM AUTHOR]

Spoligotyping and exact tandem repeat (ETR) analysis of Mycobacterium bovis and M. caprae isolated strains has been routinely carried out in Italy since 2000 to obtain a database of genetic profiles and support traditional epidemiological investigations. In this study, we characterized 1,503 M. bovis and 57 M. caprae isolates obtained from 2000 to 2006 in 747 cattle herds mainly located in northern Italy. We identified 81 spoligotypes and 113 ETR profiles, while the combination of spoligotyping/ETR analysis differentiated 228 genotypes, with genotypic diversity indices of 0.70 (spoligotyping), 0.94 (ETR-A to -E typing), and 0.97 (spoligotyping/ETR-A to -E typing), respectively. Despite the high degree of resolution obtained, the spoligotyping/ETR methods were not discriminative enough in the case of genotypes characterized by the combination of SB0120, the predominant spoligotype in Italy, with the most common ETR profiles. To obtain a more informative subset of typing loci, 24 mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) markers were evaluated by analyzing a panel of 100 epidemiologically unrelated SB0120 isolates. The panel was differentiated into 89 profiles with an overall genotypic diversity of 0.987 that could be also achieved by using a minimal group of 13 loci: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336. The allelic diversity index and the stability of single loci was evaluated to provide the most discriminative genotyping method for locally prevalent strains.