The Role of Innate Immune Response and Microbiome in Resilience of Dairy Cattle to Disease: The Mastitis Model

Animal health is affected by many factors such as metabolic stress, the immune system, and epidemiological features that interconnect. The immune system has evolved along with the phylogenetic evolution as a highly refined sensing and response system, poised to react against diverse infectious and non-infectious stressors for better survival and adaptation. It is now known that high genetic merit for milk yield is correlated with a defective control of the inflammatory response, underlying the occurrence of several production diseases. This is evident in the mastitis model where high-yielding dairy cows show high disease prevalence of the mammary gland with reduced effectiveness of the innate immune system and poor control over the inflammatory response to microbial agents. There is growing evidence of epigenetic effects on innate immunity genes underlying the response to common microbial agents. The aforementioned agents, along with other non-infectious stressors, can give rise to abnormal activation of the innate immune system, underlying serious disease conditions, and affecting milk yield. Furthermore, the microbiome also plays a role in shaping immune functions and disease resistance as a whole. Accordingly, proper modulation of the microbiome can be pivotal to successful disease control strategies. These strategies can benefit from a fundamental re-appraisal of native cattle breeds as models of disease resistance based on successful coping of both infectious and non-infectious stressors

Milk microbiome diversity and bacterial group prevalence in a comparison between healthy Holstein Friesian and Rendena cows

Dry and early lactation periods represent the most critical phases for udder health in cattle, especially in highly productive breeds, such as the Holstein Friesian (HF). On the other hand, some autochthonous cattle breeds, such as the Rendena (REN), have a lower prevalence of mastitis and other transition-related diseases. In this study, milk microbiota of 6 HF and 3 REN cows, all raised on the same farm under the same conditions, was compared. A special focus was placed on the transition period to define bacterial groups' prevalence with a plausible effect on mammary gland health. Four time points (dry-off, 1 d, 7-10 d and 30 d after calving) were considered. Through 16S rRNA sequencing, we characterized the microbiota composition for 117 out of the 144 milk samples initially collected, keeping only the healthy quarters, in order to focus on physiological microbiome changes and avoid shifts due to suspected diseases. Microbial populations were very different in the two breeds along all the time points, with REN milk showing a significantly lower microbial biodiversity. The taxonomic profiles of both cosmopolitan and local breeds were dominated by Firmicutes, mostly represented by the Streptococcus genus, although in very different proportions (HF 27.5%, REN 68.6%). Large differences in HF and REN cows were, also, evident from the metabolic predictive analysis from microbiome data. Finally, only HF milk displayed significant changes in the microbial composition along the transition period, while REN maintained a more stable microbiota. In conclusion, in addition to the influence on the final characteristics of dairy products obtained from milk of the two breeds, differences in the milk microbiome might, also, have an impact on their mammary gland health

Antegrade subinguinal sclerotization with temporary clamping of the spermatic cord: a new surgical technique for varicocele

Objectives : The purpose of our study was to evaluate the duration, effectiveness, and complications associated with a new operating technique for varicocele, using a subinguinal surgical approach and antegrade sclerotization of the spermatic veins. Methods : A total of 756 varicocele patients who came under our care for infertility underwent surgical treatment with our technique. The diagnosis was based on clinical examination and confirmed by color-Doppler ultrasound of the spermatic cord. Only patients with continuous basal reflux inside the left spermatic vein detected in orthostatism underwent operation. The Colpi technique was used, which consists of a subinguinal incision with suspension of the spermatic cord; cord clamping for 8-10 minutes using two elastic bands; and injection of 1.5-3 mL of sclerosing agent during induced ischemia without any intraoperative radiological control. Results : The average operating time was 25 minutes (range: 18-45 minutes). At the 3-month postoperative follow-up, there were 15 cases of persistent reflux (1.9%), 6 cases of hydrocele requiring surgical correction (0.7%), and 50 cases of fibrotic sequelae of penile lymphangiitis (6.6%). Conclusions : The new technique was more effective than the previous ones, with the exception of the microsurgical technique, which, however, takes 2-3 times longer to perform. The only significant complication was superficial single-vessel lymphangiitis of the penis, which resolved within 3 months with no apparent consequences. In conclusion, this new operating technique for varicocele is simpler to perform and may be effective compared with other techniques

Study of bovine milk microbiota biodiversity in autoctonous italian breeds

In order to valorize the autoctonous cows milk, a better knowledge of its quality and features is requested. In the last few years a large interest on the milk microbiota composition has raised due to the impact of the microorganisms could have on the health status of the animals, on the public health and on the quality of the dairy products. Preliminary data on mammary microbiota of the ruminant species (cow, sheep and buffalo) showed that the row milk includes a complex community of microbes affected by both endogenous and esogenous factors. The present study aims to better understand the differences of the molecular mechanisms of the microbiota interaction in the Rendena\u2019s milk compared to Holstein breed. Microbial composition of the milk from 3 Rendena cows was compared to that from 3 Holstein cows from the same farm with the same management. Quarter samples were collected from each cow at different time points: dry off (T1), at calving (T2), 7-10 days after calving (T3) and 30 days after calving (T4). We analyzed somatic cell count (SCC) and bacteriological culture for each quarter. For the microbiome analysis, the bacterial DNA was extracted from each quarter using a protocol previously described (Cremonesi et al., 2005) with some modifications and the 16S rRNA gene (V3-V4 region) was analyzed by Miseq (Illumina). Bacteriological analysis showed the absence of contagious bacteria such as Staphylococcus. aureus and Streptococcus. agalactiae in both breeds. The predominant Rendena\u2019s phylum was Firmicutes (94.3%). In Holstein the milk microbiota was more complex with Firmicutes (62.6%), Proteobacteria (19.2%), Bacteriodetes (7.5%) and Actinobacteria (6.8%). The analysis of the genera showed that in Rendena cows the main microorganisms were Streptococcus (70.7%), Lactobacillus (10.4%) and Pediococcus (5.8%), whereas in the Holstein cows were Streptococci (27.1%), Staphylococci (5.5%) and Lactobacilli (4.5%). Regarding the species of the Streptococcus genus we demontrated a significant difference between the two breeds. Indeed, in the Rendena cows the 51,6% of the present Streptococci were Streptococcus thermophilus, that in the Holstein cows represent only 1.72% of the observed Steptococci. Streptococcus thermophilus is a lactic bacterium very usefull in the dairy products (fermented milks, yogourt, different cheeses). The strong presence of Streptococcus thermophilus in the Rendena milk makes it more suitable for the dairy industry. The different microbial composition of the milk from the two breeds could also have an impact on the immune response and the infections of the udder that could help to explain the high resistance to mastitis of the autoctonous Italian cow breeds. In order to better clarify this hypothesis gene expression and proteomic analysis are in progress

Milk's microbiota and immunological traits modifies during the periparturient period in Holstein cows: possible implication on animal health and milk quality

In dairy cows the transition from late gestation to early lactation involves dramatic physiological, metabolic and immune system changes. In this period, the animals are at high risk for diseases, both metabolic and infectious. The aim of this study was to compare the microbiota and immunological/physiological parameter of the milk during the periparturient period in order to assess the possible implication on animal health and milk quality. Milk samples were collected from six pluriparous Holstein cows at dry-off (T1), day after calving (T2), 7-10 days after calving (T3), 30 days after calving (T4) and 60-70 days after calving (T5). To determine the udder health status, a standard bacteriological analysis and SCC on milk samples were performed. Microbiome analysis by Miseq (Illumina) was achieved on bacterial 16S rRNA gene. Milk proteins were evaluated by SDS-PAGE and by densitometric analysis for assessment of total protein profiles. In addition, the presence of the cathelicidin, S100A9 and lysozyme was estimated. The expression of CD45, KRT5, IL-1b and TNFa messengers was analyzed in the isolated milk cells. Finally, the expression of PTX3 and IL-1R8 transcripts in the milk fat globules was analyzed. Bacteriological analysis showed the absence of contagious bacteria such as Staph. aureus and S. agalactiae. An interesting modification of the leukocyte/exfoliated epithelial cell ratio during peripartum was observed. Despite the increased presence of leukocytes compared to the epithelial exfoliated cells in the milk at calving, we observed a reduction in proinflammatory cytokine mRNA. These data have also been integrated with information on protein markers of inflammation. Our results confirmed that the peripartum is a crucial period for dairy cows due to the physical-metabolic stresses, neuropeptides and neuroendocrine hormones changes that contribute to impair the immune system, leading to an increased susceptibility of the animals to diseases