Hygiene Assessment of Buffalo Milking Parlours in Campania Region, Italy: A Preliminary Study by Using ATP Luminometry and Bacteriological Investigation.

Careful cleaning of a milking parlour and its equipment is fundamental to guarantee good raw milk quality and prevent the dissemination of bacteria and improve animal welfare. This study aimed to investigate, using an ATP-bioluminescence assay and bacteriological analysis, the bacterial contamination of milking parlours on milking parlour surfaces of buffalo farms in the Campania Region, evaluating the seasonal dynamics during the year 2022. Eight farms were selected by the Italian ClassyFarm system, which assesses the level of animal welfare and biosecurity according to risk analysis. Before sampling, all dairy farm owners filled out a questionnaire on milking management, animal hygiene, and health. The questionnaires evidenced similar cleaning procedures but an absence of a standardised cleaning protocol among the different farms. ATP bioluminescence results evidenced similar levels of contamination in all the selected buffalo farms, and the season comparison showed no significant differences. A variation in the percentages of bacterial isolates during the different seasons was observed, with a higher prevalence of Enterobacteriaceae (38%) in summer. A small number of samples exhibited an absence of bacterial growth. Identifying bacteria is crucial for understanding the microorganisms present in the milking parlour, yet employing ATP luminometry could offer broad and accurate applications in buffalo milking parlours. In conclusion, the use of ATP bioluminescence for evaluating the hygiene of a buffalo milking parlour could represent a further important advancement in dairy farming technology.

In vivo biological validation of in silico analysis: A novel approach for predicting the effects of TLR4 exon 3 polymorphisms on brucellosis.

The role of the Toll-like receptor 4 (TLR4) is of recognising intracellular and extracellular pathogens and of activating the immune response. This process can be compromised by single nucleotide polymorphisms (SNPs) which might affect the activity of several TLRs. The aim of this study is of ascertaining whether SNPs in the TLR4 of Bubalus bubalis infected by Brucella abortus, compromise the protein functionality. For this purpose, a computational analysis was performed. Next, computational predictions were confirmed by performing genotyping analysis. Finally, NMR-based metabolomics analysis was performed to identify potential biomarkers for brucellosis. The results indicate two SNPs (c. 672 A > C and c. 902 G > C) as risk factor for brucellosis in Bubalus bubalis, and three metabolites (lactate, 3-hydroxybutyrate and acetate) as biological markers for predicting the risk of developing the disease. These metabolites, together with TLR4 structural modifications in the MD2 interaction domain, are a clear signature of the immune system alteration during diverse Gram-negative bacterial infections. This suggests the possibility to extend this study to other pathogens, including Mycobacterium tuberculosis. In conclusion, this study combines multidisciplinary approaches to evaluate the biological and structural effects of SNPs on protein function.

Adaptation of perennial flowering phenology across the European range of Arabis alpina.

Flowering phenology is important in the adaptation of many plants to their local environment, but its adaptive value has not been extensively studied in herbaceous perennials. We used Arabis alpina as a model system to determine the importance of flowering phenology to fitness of a herbaceous perennial with a wide geographical range. Individual plants representative of local genetic diversity (accessions) were collected across Europe, including in Spain, the Alps and Scandinavia. The flowering behaviour of these accessions was documented in controlled conditions, in common-garden experiments at native sites and in situ in natural populations. Accessions from the Alps and Scandinavia varied in whether they required exposure to cold (vernalization) to induce flowering, and in the timing and duration of flowering. By contrast, all Spanish accessions obligately required vernalization and had a short duration of flowering. Using experimental gardens at native sites, we show that an obligate requirement for vernalization increases survival in Spain. Based on our analyses of genetic diversity and flowering behaviour across Europe, we propose that in the model herbaceous perennial A. alpina, an obligate requirement for vernalization, which is correlated with short duration of flowering, is favoured by selection in Spain where the plants experience a long growing season.

Sequencing and Characterization of αs2-Casein Gene (CSN1S2) in the Old-World Camels Have Proven Genetic Variations Useful for the Understanding of Species Diversification.

The CSN1S2 gene encodes αs2-casein, the third most abundant protein in camel milk. Despite its importance in foals, human nutrition, and dairy processing, the CSN1S2 gene in camels has received little attention. This study presents the first complete characterization of the CSN1S2 gene sequence in Old-World camels (Camelus bactrianus and Camelus dromedarius). Additionally, the gene promoter, consisting of 752 bp upstream of exon 1, was analyzed. The entire gene comprises 17 exons, ranging in length from 24 bp (exons 4, 8, 11, and 13) to 280 bp (exon 17). Interesting was the identification of the exon 12 in both species. The promoter analysis revealed 24 putative binding sites in the Bactrian camel and 22 in dromedary camel. Most of these sites were typical elements associated with milk protein, such as C/EBP-α, C/EBP-ß, Oct-1, and AP1. The SNP discovery showed relatively high genetic diversity compared to other camel casein genes (CSN1S1, CSN2, and CSN3), with a total of 34 polymorphic sites across the two species. Particularly noteworthy is the transition g.311G>A in the CSN1S2 promoter, creating a new putative consensus binding site for a C/EBP-ß in the Bactrian camel. At the exon level, two novel variants were found. One was detected in exon 6 of the Bactrian camel (g.3639C>G), resulting in an amino acid replacement, p.36Ile>Met. The second variant was found in noncoding exon 17 of dromedary CSN1S2 (g.1511G>T). Although this mutation occurs in the 3'-UnTranslated Region, it represents the first example of exonic polymorphism in the CSN1S2 for this species. This SNP also affects the binding sites of different microRNAs, including the seed sequence of the miRNA 4662a-3p, highlighting its role as a regulatory factor for CSN1S2 gene. A PCR-RFLP was set up for genotyping a dromedary Tunisian population (n = 157), and the minor allele frequency was found to be 0.27 for the G allele, indicating a potential yield improvement margin. The interspersed elements (INEs) analysis revealed 10 INEs covering 7.34% and 8.14% of the CSN1S2 sequence in the Bactrian and dromedary camels, respectively. Furthermore, six elements (A, B, F, H, I, and L) are shared among cattle and camels and are partially found in other ruminants, suggesting a common ancestral origin of these retrotransposons. Conversely, elements C, D, E, and G are specific to camels.

The Safety of Ethnic Foods in Campania Region: A Preliminary Study.

Ethnic food is produced by an ethnic group-using their familiarity with local ingredients of plants and/or animal origin-and it is consumed in a country other than the country of origin. In Italy, the ethnic food market has expanded over the last three decades. The aim of this study was to evaluate the correct labeling, the microbiological communities and the allergens present in 50 ethnic foods. The visual inspection of labels and microbiological and allergen analyses have been carried out for evaluating their food safety. The visual inspection of labels revealed the absence of labeling in Italian and/or a failure to specify the place of origin. Microbiological analyses showed the absence of pathogens (i.e., Salmonella spp., Listeria monocytogenes and E. coli 0157:H7) in all matrices, but the presence of process hygiene indicator bacteria (total bacterial count, Coagulase-positive Staphylococci, Bacillus cereus, coliforms, yeasts and molds) was found in 37 samples. With regard to allergens, 12 samples were non-compliant for the presence of at least one allergen, while only two products were of species different from those declared on the label. This research highlights the need to increase the control of ethnic foods and also to improve the labeling system by standardizing international regulations.

Genomic Basis of Adaptation to a Novel Precipitation Regime.

Energy production and metabolism are intimately linked to ecological and environmental constraints across the tree of life. In plants, which depend on sunlight to produce energy, the link between primary metabolism and the environment is especially strong. By governing CO2 uptake for photosynthesis and transpiration, leaf pores, or stomata, couple energy metabolism to the environment and determine productivity and water-use efficiency (WUE). Although evolution is known to tune physiological traits to the local environment, we lack knowledge of the specific links between molecular and evolutionary mechanisms that shape this process in nature. Here, we investigate the evolution of stomatal conductance and WUE in an Arabidopsis population that colonized an island with a montane cloud scrubland ecosystem characterized by seasonal drought and fog-based precipitation. We find that stomatal conductance increases and WUE decreases in the colonizing population relative to its closest outgroup population from temperate North Africa. Genome-wide association mapping reveals a polygenic basis of trait variation, with a substantial contribution from a nonsynonymous single-nucleotide polymorphism in MAP KINASE 12 (MPK12 G53R), which explains 35% of the phenotypic variance in WUE in the island population. We reconstruct the spatially explicit evolutionary history of MPK12 53R on the island and find that this allele increased in frequency in the population due to positive selection as Arabidopsis expanded into the harsher regions of the island. Overall, these findings show how adaptation shaped quantitative eco-physiological traits in a new precipitation regime defined by low rainfall and high humidity.

A two-step adaptive walk rewires nutrient transport in a challenging edaphic environment.

Most well-characterized cases of adaptation involve single genetic loci. Theory suggests that multilocus adaptive walks should be common, but these are challenging to identify in natural populations. Here, we combine trait mapping with population genetic modeling to show that a two-step process rewired nutrient homeostasis in a population of Arabidopsis as it colonized the base of an active stratovolcano characterized by extremely low soil manganese (Mn). First, a variant that disrupted the primary iron (Fe) uptake transporter gene (IRT1) swept quickly to fixation in a hard selective sweep, increasing Mn but limiting Fe in the leaves. Second, multiple independent tandem duplications occurred at NRAMP1 and together rose to near fixation in the island population, compensating the loss of IRT1 by improving Fe homeostasis. This study provides a clear case of a multilocus adaptive walk and reveals how genetic variants reshaped a phenotype and spread over space and time.

Parallel reduction in flowering time from de novo mutations enable evolutionary rescue in colonizing lineages.

Understanding how populations adapt to abrupt environmental change is necessary to predict responses to future challenges, but identifying specific adaptive variants, quantifying their responses to selection and reconstructing their detailed histories is challenging in natural populations. Here, we use Arabidopsis from the Cape Verde Islands as a model to investigate the mechanisms of adaptation after a sudden shift to a more arid climate. We find genome-wide evidence of adaptation after a multivariate change in selection pressures. In particular, time to flowering is reduced in parallel across islands, substantially increasing fitness. This change is mediated by convergent de novo loss of function of two core flowering time genes: FRI on one island and FLC on the other. Evolutionary reconstructions reveal a case where expansion of the new populations coincided with the emergence and proliferation of these variants, consistent with models of rapid adaptation and evolutionary rescue.

Impact of Freezing on the Microbiological Quality and Physical Characteristics of Buffalo Mozzarella Cheese.

Buffalo Mozzarella cheese from Campania is one of the most worldwide appreciated Italian dairy products. The increased demand for buffalo dairy products and the limited availability of the finest buffalo milk has prompted the diffusion of illicit practices, such as the use of milk, curd, or other products that are frozen or bought at low cost. The aim of this research was to provide preliminary results about the trend of the microbial communities of buffalo milk, curd and Buffalo Mozzarella cheese, during freezing storage of eleven months. At the same time, the alterations of physical properties and the presence of the molecular marker "γ4-casein", have been investigated. The results showed that freezing reduced the concentrations of the total bacterial count, Enterobacteriaceae, coliforms, Escherichia coli and yeasts in fresh milk and, the concentrations of the total bacterial count, coliforms, lactic acid bacteria and yeasts in mature curd. In the finished product, no notable decreases were observed, except for lactic acid bacteria. About the γ4-casein, no increase was observed in all matrices. These preliminary results allow us to conclude that the freezing process if properly carried out, does not compromise the microbiological quality and the physical properties of the Buffalo Mozzarella cheese.

Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese.

Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.

A Review of Hemp as Food and Nutritional Supplement.

The term "hemp" refers to Cannabis sativa cultivars grown for industrial purposes that are characterized by lower levels of tetrahydrocannabinol (THC), the active principle responsible for Cannabis psychotropic effects. Hemp is an extraordinary crop, with enormous social and economic value, since it can be used to produce food, textiles, clothing, biodegradable plastics, paper, paint, biofuel, and animal feed, as well as lighting oil. Various parts of the hemp plant represent a valuable source of food and ingredients for nutritional supplements. While hemp inflorescence is rich in nonpsychoactive, yet biologically active cannabinoids, such as cannabidiol (CBD), which exerts potent anxiolytic, spasmolytic, as well as anticonvulsant effects, hempseed has a pleasant nutty taste and represents a valuable source of essential amino acids and fatty acids, minerals, vitamins, and fibers. In addition, hempseed oil is a source of healthy polyunsaturated fatty acids, and hemp sprouts are rich in antioxidants. This review article aims to provide a comprehensive outlook from a multidisciplinary perspective on the scientific evidence supporting hemp beneficial properties when consumed as food or supplement. Marketing of hemp-derived products is subjected to diversified and complex regulations worldwide for several reasons, including the fact that CBD is also the active principal of pharmaceutical agents and that regulatory bodies in some cases ban Cannabis inflorescence regardless of its THC content. Some key regulatory aspects of such a complex scenario are also analyzed and discussed in this review article.

A New Catalog of Structural Variants in 1,301 A. thaliana Lines from Africa, Eurasia, and North America Reveals a Signature of Balancing Selection at Defense Response Genes.

Genomic variation in the model plant Arabidopsis thaliana has been extensively used to understand evolutionary processes in natural populations, mainly focusing on single-nucleotide polymorphisms. Conversely, structural variation has been largely ignored in spite of its potential to dramatically affect phenotype. Here, we identify 155,440 indels and structural variants ranging in size from 1 bp to 10 kb, including presence/absence variants (PAVs), inversions, and tandem duplications in 1,301 A. thaliana natural accessions from Morocco, Madeira, Europe, Asia, and North America. We show evidence for strong purifying selection on PAVs in genes, in particular for housekeeping genes and homeobox genes, and we find that PAVs are concentrated in defense-related genes (R-genes, secondary metabolites) and F-box genes. This implies the presence of a "core" genome underlying basic cellular processes and a "flexible" genome that includes genes that may be important in spatially or temporally varying selection. Further, we find an excess of intermediate frequency PAVs in defense response genes in nearly all populations studied, consistent with a history of balancing selection on this class of genes. Finally, we find that PAVs in genes involved in the cold requirement for flowering (vernalization) and drought response are strongly associated with temperature at the sites of origin.

The Union Is Strength: The Synergic Action of Long Fatty Acids and a Bacteriophage against Xanthomonas campestris Biofilm.

Xanthomonas campestris pv. campestris is known as the causative agent of black rot disease, which attacks mainly crucifers, severely lowering their global productivity. One of the main virulence factors of this pathogen is its capability to penetrate and form biofilm structures in the xylem vessels. The discovery of novel approaches to crop disease management is urgent and a possible treatment could be aimed at the eradication of biofilm, although anti-biofilm approaches in agricultural microbiology are still rare. Considering the multifactorial nature of biofilm, an effective approach against Xanthomonas campestris implies the use of a multi-targeted or combinatorial strategy. In this paper, an anti-biofilm strategy based on the use of fatty acids and the bacteriophage (Xccφ1)-hydroxyapatite complex was optimized against Xanthomonas campestris mature biofilm. The synergic action of these elements was demonstrated and the efficient removal of Xanthomonas campestris mature biofilm was also proven in a flow cell system, making the proposed approach an effective solution to enhance plant survival in Xanthomonas campestris infections. Moreover, the molecular mechanisms responsible for the efficacy of the proposed treatment were explored.

Interaction between MyD88, TIRAP and IL1RL1 against Helicobacter pylori infection.

The Toll-interleukin 1 receptor superfamily includes the genes interleukin 1 receptor-like 1 (IL1RL1), Toll like receptors (TLRs), myeloid differentiation primary-response 88 (MyD88), and MyD88 adaptor-like (TIRAP). This study describes the interaction between MyD88, TIRAP and IL1RL1 against Helicobacter pylori infection. Cases and controls were genotyped at the polymorphic sites MyD88 rs6853, TIRAP rs8177374 and IL1RL1 rs11123923. The results show that specific combinations of IL1RL1-TIRAP (AA-CT; P: 2,8 × 10-17) and MyD88-TIRAP-IL1RL1 (AA-CT-AA; P: 1,4 × 10-8) - but not MyD88 alone-act synergistically against Helicobacter pylori. Nuclear magnetic resonance (NMR) clearly discriminates cases from controls by highlighting significantly different expression levels of several metabolites (tyrosine, tryptophan, phenylalanine, branched-chain amino acids, short chain fatty acids, glucose, sucrose, urea, etc.). NMR also identifies the following dysregulated metabolic pathways associated to Helicobacter pylori infection: phenylalanine and tyrosine metabolism, pterine biosynthesis, starch and sucrose metabolism, and galactose metabolism. Furthermore, NMR discriminates between the cases heterozygous at the IL1RL1 locus from those homozygous at the same locus. Heterozygous patients are characterized by high levels of lactate, and IL1RL1-both associated with anti-inflammatory activity-and low levels of the pro-inflammatory molecules IL-1ß, TNF-α, COX-2, and IL-6.

An Innovative Approach to Control H. pylori-Induced Persistent Inflammation and Colonization.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium which colonizes the human stomach. The ability of H. pylori to evade the host defense system and the emergence of antibiotic resistant strains result in bacteria persistence and chronic inflammation, which leads to both severe gastric and extra-gastric diseases. Consequently, innovative approaches able to overcome H. pylori clinical outcomes are needed. In this work, we develop a novel non-toxic therapy based on the synergistic action of H. pylori phage and lactoferrin adsorbed on hydroxyapatite nanoparticles, which effectively impairs bacteria colonization and minimizes the damage of the host pro-inflammatory response.

Plant Dynamic Metabolic Response to Bacteriophage Treatment After Xanthomonas campestris pv. campestris Infection.

Periodic epidemics of black rot disease occur worldwide causing substantial yield losses. Xanthomonas campestris pv. campestris (Xcc) represents one of the most common bacteria able to cause the above disease in cruciferous plants such as broccoli, cabbage, cauliflower, and Arabidopsis thaliana. In agriculture, several strategies are being developed to contain the Xanthomonas infection. The use of bacteriophages could represent a valid and efficient approach to overcome this widespread phenomenon. Several studies have highlighted the potential usefulness of implementing phage therapy to control plant diseases as well as Xcc infection. In the present study, we characterized the effect of a lytic phage on the plant Brassica oleracea var. gongylodes infected with Xcc and, for the first time, the correlated plant metabolic response. The results highlighted the potential benefits of bacteriophages: reduction of bacterium proliferation, alteration of the biofilm structure and/or modulation of the plant metabolism and defense response.

Bacteriophages Promote Metabolic Changes in Bacteria Biofilm.

Bacterial biofilm provides bacteria with resistance and protection against conventional antimicrobial agents and the host immune system. Bacteriophages are known to move across the biofilm to make it permeable to antimicrobials. Mineral hydroxyapatite (HA) can improve the lytic activity of bacteriophages, and, together with eicosanoic acid (C20:0), can destroy the biofilm structure. Here, we demonstrate the efficacy of the combined use of phage, HA and C20:0 against Xanthomonas campestris pv campestris (Xcc) biofilm. We used nuclear magnetic resonance (NMR)-based metabolomics to investigate the molecular determinants related to the lytic action, aiming at identifying the metabolic pathways dysregulated by phage treatment. Furthermore, we identified specific markers (amino acids, lactate and galactomannan) which are involved in the control of biofilm stability. Our data show that Xccφ1, alone or in combination with HA and C20:0, interferes with the metabolic pathways involved in biofilm formation. The approach described here might be extended to other biofilm-producing bacteria.

Antibiofilm Activity of a Trichoderma Metabolite against Xanthomonas campestris pv. campestris, Alone and in Association with a Phage.

Biofilm protects bacteria against the host's immune system and adverse environmental conditions. Several studies highlight the efficacy of lytic phages in the prevention and eradication of bacterial biofilms. In this study, the lytic activity of Xccφ1 (Xanthomonas campestris pv. campestris-specific phage) was evaluated in combination with 6-pentyl-α-pyrone (a secondary metabolite produced by Trichoderma atroviride P1) and the mineral hydroxyapatite. Then, the antibiofilm activity of this interaction, called a φHA6PP complex, was investigated using confocal laser microscopy under static and dynamic conditions. Additionally, the mechanism used by the complex to modulate the genes (rpf, gumB, clp and manA) involved in the biofilm formation and stability was also studied. Our results demonstrated that Xccφ1, alone or in combination with 6PP and HA, interfered with the gene pathways involved in the formation of biofilm. This approach can be used as a model for other biofilm-producing bacteria.

Screen Printed Based Impedimetric Immunosensor for Rapid Detection of Escherichia coli in Drinking Water.

The development of a simple and low cost electrochemical impedance immunosensor based on screen printed gold electrode for rapid detection of Escherichia coli in water is reported. The immunosensor is fabricated by immobilizing anti-E. coli antibodies onto a gold surface in a covalent way by the photochemical immobilization technique, a simple procedure able to bind antibodies upright onto gold surfaces. Impedance spectra are recorded in 0.01 M phosphate buffer solution (PBS) containing 10 mM Fe(CN)63-/Fe(CN)64- as redox probe. The Nyquist plots can be modelled with a modified Randles circuit, identifying the charge transfer resistance Rct as the relevant parameter after the immobilization of antibodies, the blocking with BSA and the binding of E. coli. The introduction of a standard amplification procedure leads to a significant enhancement of the impedance increase, which allows one to measure E. coli in drinking water with a limit of detection of 3 × 101 CFU mL-1 while preserving the rapidity of the method that requires only 1 h to provide a "yes/no" response. Additionally, by applying the Langmuir adsorption model, we are able to describe the change of Rct in terms of the "effective" electrode, which is modified by the detection of the analyte whose microscopic conducting properties can be quantified.

Identification and Characterization of Nasal Polyposis and Mycoplasma Superinfection by Scanning Electron Microscopy and Nasal Cytology with Optical Microscopy: A Case Report.

Nasal polyposis is characterized by benign, non-cancerous and painless growths originating in the tissue of the nasal cavities and paranasal sinuses. Polyps arise from chronic inflammation due to asthma, recurrent infections, allergies, drug sensitivity or immune disorders. They can obstruct the nasal cavities and thus cause respiratory problems, a reduction in the sense of smell and susceptibility to infections. Furthermore, nasal polyps can recur. Hence the importance of using valid diagnostic methods. In this work, the diagnostic investigation carried out by scanning electron microscopy (SEM) and nasal cytology led, for the first time, to the identification of a mycoplasma superinfection on nasal polyposis.