Milk Somatic Cell Count and Polymorphonuclear Cells in Healthy Quarters of Cows That Underwent Blanket and Selective Dry Therapy: An Italian Case Study.

The incidence of mastitis increases with parity in dairy cattle and multiparous cows are often treated at drying off to limit the risk of udder health issues and support mammary gland tissues recovery. Milk somatic cells count (SCC, cells/mL) comprises different white blood cells fractions and is worldwide used to monitor and genetically improve udder health. Nevertheless, only certain SCC fractions increase when an udder inflammation occurs. Considering that antibiotic use for preventive purposes will be forbidden in 2022, we compared two different dry therapy protocols, blanket (BDCT) and selective (SDCT), on different SCC fractions in healthy quarters milk. Multiparous Holstein cows were enrolled in a randomized controlled trial and SCC, neutrophils, macrophages, lymphocytes, polymorphonuclear cells (PMN) and differential somatic cell count (DSCC) recorded after the experimental drying off were available. Significant differences were observed between the two protocols, with more favorable parameters in BDCT than SDCT cows. Results showed that moving from BDCT to SDCT is expected to significantly increase some SCC fractions, such as PMN, in healthy quarters. The baseline SCC level at the onset of lactation was greater in cows under SDCT than BDCT. Although not significant, clinical mastitis prevalence was numerically lower in BDCT (7.32%) than SDCT (8.62%). In this study we referred to a limited number of cows, but still findings will be useful to improve the knowledge on the impact of SDCT on milk SCC fractions in healthy quarters.

Application of the Micro Biological Survey analytical method for the determination of bacterial load in cow raw milk.

The aim of this study was to evaluate the performance of "Micro Biological Survey - MBS Test" in the enumeration of bacterial load in cow raw milk. The MBS test is based on a colorimetric method recently developed and patented by "Roma Tre" University, Italy. The evaluation of the performance of the MBS method was carried out by comparison with plate count at 30°C (gold standard) and flow cytometry. Thirteen independent set of experiments were performed analyzing a total of 104 samples of cow raw milk with the selected methods. Results obtained using the MBS method are comparable with those obtained with the plate count method at 30°C (CFU/mL) and flow cytometry technology; in particular, the results obtained with the MBS method are very close to plate count's at 30°C. On the other hand, there are statistically significant differences between these two methods' and flow cytometry technology's results that could be due to the different experimental conditions.

On the Effect of the Temperature-Humidity Index on Buffalo Bulk Milk Composition and Coagulation Traits.

Little is known about the effects of high levels of environmental temperature and humidity on milk yield and quality in buffaloes since this species is known to be more heat tolerant than cattle. However, the distribution of sweat glands and the dark skin color can negatively affect heat tolerance. Moreover, due to increased global temperatures, concerns regarding heat stress and thermoregulation in dairy animals, including buffaloes, have been extended to the northern hemisphere. In this study, the effects of both the temperature-humidity index (THI) and the maximum daily temperature-humidity index (MTHI) were estimated on bulk milk traits, namely fat, protein, lactose, urea content, pH levels, somatic cell score, coagulation properties, and bacteria count. The dataset consisted of repeated data from 99 Mediterranean water buffalo farms, and mixed models were used for the analyses. Supporting the negative correlations observed, bulk milk fat, protein, and lactose content were significantly lower when THI and MTHI were higher. Similarly, milk pH was lower when THI and MTHI were high; however, high levels of THI or MTHI seemed to not be markedly associated with the milk's coagulation ability. According to both analysis of variance and correlations, the somatic cell score was not significantly affected by the THI and MTHI. This is the first study based on a large dataset that evaluates the impact of high temperature and humidity in Italian buffalo milk and that provides correlations with traits of interest for the dairy industry, i.e., milk acidity and coagulation ability. In general, findings show that the effects of elevated THI and heat stress on bulk milk quality in buffalo is less evident than in cattle. These preliminary results intend to open debate on the issue of heat stress in dairy buffaloes that are reared in temperate regions. Further studies should focus on individual milk and performance and should investigate the relationship between high THI and buffalo fertility, behavior, and welfare.

Rapid Microbiological Assessment in Raw Milk: Validation of a Rapid Alternative Method for the Assessment of Microbiological Quality in Raw Milk.

The consumption of dairy products and the dairy industry are one of the main global agri-food sectors for its size, economic importance, and level of technology. Microbiological quality of pasteurized milk or other milk products is dependent on microbiological quality of raw milk. A variety of microbiological count methods is available for monitoring the hygienic quality of raw milk. Among them, the pour plate method is the official essay for counting the number of colony-forming units in milk samples according to International Organization for Standardization (ISO) No. 4833-1:2013. The aim of the present study is the validation of the Micro Biological Survey (MBS) method, against the reference plate-count method, for the assessment of the microbiological quality of raw milk. This comparative study, performed in collaboration with the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri (IZSLT), demonstrates the accuracy of this alternative method for the determination of total viable bacterial count in cow's raw milk. The results obtained with the MBS method highlight its potential as a valid tool for reliable microbiological analysis in dairy industries.

An Evaluation of the Clotting Properties of Three Plant Rennets in the Milks of Different Animal Species.

Cynara cardunculus, Carica papaya and Ficus carica extracts are proposed as milk coagulants herein. Their coagulation efficiency was measured in bovine, buffalo, goat and sheep milk incubated at different temperatures. The milk-clotting and proteolytic activities as well as the lactodynamographic parameters were determined considering animal rennet as a reference coagulant. The vegetable coagulant, extracted from C. cardunculus pistils, proved to be the most suitable milk-clotting enzyme for cheesemaking, since it possesses similar milk clotting properties to conventional calf rennet. F. carica latex, but seemed to be a promising alternative coagulant at higher temperatures. The strong proteolytic activity of papain caused poor milk coagulation in all milk samples. To conclude, this result also supports the original hypothesis of this study that the excessive proteolytic nature of plant coagulants can negatively affect the cheesemaking process. The optimization of using a plant rennet in a dairy application can be done by selecting the appropriate plant rennet with a consistent clotting efficiency. These innovative manufacturing processes may also lead to the optimization and production of new cheese varieties.

From Invention to Innovation: Risk Analysis to Integrate One Health Technology in the Dairy Farm.

Current Hazard Analysis Critical Control Points (HACCP) approaches mainly fit for food industry, while their application in primary food production is still rudimentary. The European food safety framework calls for science-based support to the primary producers' mandate for legal, scientific, and ethical responsibility in food supply. The multidisciplinary and interdisciplinary project ALERT pivots on the development of the technological invention (BEST platform) and application of its measurable (bio)markers-as well as scientific advances in risk analysis-at strategic points of the milk chain for time and cost-effective early identification of unwanted and/or unexpected events of both microbiological and toxicological nature. Health-oriented innovation is complex and subject to multiple variables. Through field activities in a dairy farm in central Italy, we explored individual components of the dairy farm system to overcome concrete challenges for the application of translational science in real life and (veterinary) public health. Based on an HACCP-like approach in animal production, the farm characterization focused on points of particular attention (POPAs) and critical control points to draw a farm management decision tree under the One Health view (environment, animal health, food safety). The analysis was based on the integrated use of checklists (environment; agricultural and zootechnical practices; animal health and welfare) and laboratory analyses of well water, feed and silage, individual fecal samples, and bulk milk. The understanding of complex systems is a condition to accomplish true innovation through new technologies. BEST is a detection and monitoring system in support of production security, quality and safety: a grid of its (bio)markers can find direct application in critical points for early identification of potential hazards or anomalies. The HACCP-like self-monitoring in primary production is feasible, as well as the biomonitoring of live food producing animals as sentinel population for One Health.

Molecular Epidemiology of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus in the Ovine Dairy Chain and in Farm-Related Humans.

Staphylococcus aureus is a major cause of clinical infections in humans and its enterotoxins cause foodborne disease. In the present study, we tested a total of 51 isolates of S. aureus from small-ruminant dairy farms with artisan dairy facilities, all located in Latium, Italy. The farms have a known history of a high prevalence of methicillin-resistant S. aureus (MRSA). Most of the MRSA isolates (27 of 51) belonged to spa-type t127 (43.1%), followed by t2678 (3.9%), t044 (2%), t1166 (2%), and t1773 (2%). PFGE performed on mecA positive strains identified one cluster (≥ 80% of similarity), comprising 22 MRSA. Nine of twenty-two MRSA isolates were assigned human host origin, and 13 isolates did not belong to a specific host. During the characterization study, one strain isolated from bulk tank milk samples harbored the pvl gene; the strain was not enterotoxigenic with a non-specific host according to the biotyping scheme, highlighting the possible emerging risk of transmission of bacterial virulence factors by foods, the environment, and foodhandlers. These findings stress the importance of hygienic measures at all processing steps of the food production chain and underline that monitoring for the presence of MRSA throughout the food chain is essential for public health.

Human campylobacteriosis related to the consumption of raw milk sold by vending machines in Italy: Quantitative risk assessment based on official controls over four years.

A quantitative risk assessment (RA) model was developed to describe the risk of campylobacteriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment was based on the official microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. Two separate RA models were developed, one for the consumption of boiled milk and the other for the consumption of raw milk, and two different dose-response (D-R) relationships were considered. The RA model predicted no human campylobacteriosis cases per year either in the best (4°C) storage conditions or in the case of thermal abuse in case of boiling raw milk, whereas in case of raw milk consumption the annual estimated campylobacteriosis cases depend on the dose-response relationships used in the model (D-R I or D-R II), the milk time-temperature storage conditions, consumer behaviour and age of consumers, namely young (with two cut-off values of ≤5 or ≤6 years old for the sensitive population) versus adult consumers. The annual estimated cases for young consumers using D-R II for the sensitive population (≤5 years old) ranged between 1013.7/100,000 population and 8110.3/100,000 population and for adult consumers using D-R I between 79.4/100,000 population and 333.1/100,000 population. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective and the proposed RA model represents a useful and flexible tool to perform future RAs based on local consumer habits to support decision-making on safety policies. Further educational programmes for raw milk consumers or potential raw milk consumers are required to encourage consumers to boil milk to reduce the associated risk of illness.

Quantitative risk assessment of human salmonellosis and listeriosis related to the consumption of raw milk in Italy.

Two quantitative risk assessment (RA) models were developed to describe the risk of salmonellosis and listeriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment considered the official microbiological records monitoring raw milk samples from vending machines performed by the regional veterinary authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, and consumption preference. Two separate RA models were developed: one for the consumption of boiled milk and the other for the consumption of raw milk. The RA models predicted no human listeriosis cases per year either in the best or worst storage conditions and with or without boiling raw milk, whereas the annual estimated cases of salmonellosis depend on the dose-response relationships used in the model, the milk storage conditions, and consumer behavior in relation to boiling raw milk or not. For example, the estimated salmonellosis cases ranged from no expected cases, assuming that the entire population boiled milk before consumption, to a maximum of 980,128 cases, assuming that the entire population drank raw milk without boiling, in the worst milk storage conditions, and with the lowest dose-response model. The findings of this study clearly show how consumer behavior could affect the probability and number of salmonellosis cases and in general, the risk of illness. Hence, the proposed RA models emphasize yet again that boiling milk before drinking is a simple yet effective tool to protect consumers against the risk of illness inherent in the consumption of raw milk. The models may also offer risk managers a useful tool to identify or implement appropriate measures to control the risk of acquiring foodborne pathogens. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective.

Antimicrobial Activity of Essential Oils Against Staphylococcus aureus in Fresh Sheep Cheese.

Essential oils (EOs) are aromatic oily liquids extracted from different parts of specific plants, well known especially for their aromatic and antibacterial properties. Nowadays, EOs are exploited in the food sector mainly for their aromatic properties. Thanks to their antimicrobial activity, however, they could also be used as additives to increase the safety and the shelf-life of food products. Aim of this study was to assess the antimicrobial activity of Thymus vulgaris L. oil and of Origanum vulgare L. oil against Staphylococcus aureus both in vitro and on fresh cheese, and to determine whether the use of EOs can modify the microbiological and/or chemical-physical properties of the products. The antimicrobial activity against S. aureus in vitro was assessed by preparation of the aromatogram (diffusion in agar test), minimum inhibitory concentration test and minimum bactericidal concentration assessment. Raw sheep milk was experimentally contaminated with a strain of S. aureus ATCC 25922 and was used to produce three types of fresh cheese: without EOs, with thyme and oregano EOs (both EOs at a concentration of 1:1000). The samples were analysed on the day of production, after three and seven days. The results obtained from the tests showed that the concentration of S. aureus and the counts of lactic flora remained unchanged for all types of cheese. Even the chemical-physical parameters were constant. The results of inhibition tests on the cheese disagree with those relating to the in vitro tests. Most likely this is due to the ability of EOs to disperse in the lipids the food: the higher the fat content is, the lower the oil fraction will be able to exert the antimicrobial activity.

Sheep Milk Yogurt from a Short Food Supply Chain: Study of the Microbiological, Chemico-Physical and Organoleptic Parameters in Relation to Shelf-Life.

Aim of this work was to analyse some microbiological, chemico-physical and organoleptic parameters of sheep milk yogurt during and after its declared shelf-life. Five samples of a sheep's milk yogurt of the same lot, collected from a short supply chain ovine dairy farm of the Roman province, were analysed. Declared shelf-life of the product was 30 days. The products were examined at 2, 14, 30, 35 and 40 days from the production date, performing the following microbiological analyses: enumeration of i) colony-forming units characteristic of the yogurt, ii) Enterobacteriaceae, iii) yeasts and/or moulds at 25°C. Microbiological identification was performed by miniature biochemical tests and for the lactic acid bacteria also by PCR. At every test interval, evaluation of organoleptic parameters and pH was also performed. The analysed product maintained an almost constant amount of lactic acid bacteria until the end of the declared shelf-life. Concerning lactic acid bacteria, a 100% concordance of the results observed by using biochemical identification methods and PCR assays was obtained. After 14 days from the production, the presence of yeasts (Candida famata) was revealed, while the presence of moulds was detected after 30 days. Ralstonia picketii, an environmental microorganism, was also isolated. The results obtained in this study indicate that yogurt spoilage is mainly due to the growth of specific microorganisms of spoilage, such as yeasts and moulds.

Four-year monitoring of foodborne pathogens in raw milk sold by vending machines in Italy.

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian regions (60,907 samples from 1,239 vending machines) from 2008 to 2011. Data from samples analyzed by both culture-based and real-time PCR methods were collected in one region. One hundred raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred seventy-eight of 60,907 samples were positive for one of the four foodborne pathogens investigated: 18 samples were positive for Salmonella, 83 for L. monocytogenes, 24 for E. coli O157:H7, and 53 for C. jejuni in the seven regions investigated. No significant differences in prevalence were found among regions, but a significant increase in C. jejuni prevalence was observed over the years of the study. A comparison of the two analysis methods revealed that real-time PCR was 2.71 to 9.40 times more sensitive than the culture-based method. Data on consumer habits revealed that some behaviors may enhance the risk of infection linked to raw milk consumption: 37% of consumers did not boil milk before consumption, 93% never used an insulated bag to transport raw milk home, and raw milk was consumed by children younger than 5 years of age. These results emphasize that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits will be useful for the development of a national quantitative risk assessment of Salmonella, L. monocytogenes, E. coli O157, and C. jejuni infection associated with raw milk consumption.