Frequency of non-aureus staphylococci and mammaliicocci species isolated from quarter clinical mastitis: a six-year retrospective study.

Non-aureus staphylococci and mammaliicocci (NASM) are the most frequently isolated bacterial group from bovine milk samples. Most studies focus on subclinical mastitis caused by NASM, however NASM can cause clinical mastitis (CM) as well. We evaluated retrospective data from 6 years (2017-2022) to determine the species and frequency of NASM isolated from quarter bovine CM. The data comprised of microbiological results from quarter CM samples routinely submitted to Quality Milk Production Services (QMPS), Cornell University, NY, US, for microbial identification by MALDI-TOF MS. A total of 9,909 microbiological results from 410 dairy herds were evaluated. Our results showed that 29 distinct NASM species were identified, with the 8 most prevalent NASM species being Staphylococcus chromogenes, S. haemolyticus, S. simulans, S. epidermidis, S. sciuri (now Mammaliicoccus sciuri), S. agnetis/S. hyicus, S. borealis, and S. xylosus. The NASM distribution remained similar among seasons, but the frequency of NASM CM cases was higher during the summer. Our results showed different patterns of variations in the isolation frequency over time, depending on the bacterial species: increasing or decreasing trends, cyclic fluctuations, and except for S. borealis, a significant seasonality effect for our study's most prevalent NASM was observed. This study showed that S. chromogenes remains the most frequent (43%) NASM species identified from bovine CM, followed by S. haemolyticus (18%), and S. simulans (12%).

Comparative characterisation of human and ovine non-aureus staphylococci isolated in Sardinia (Italy) for antimicrobial susceptibility profiles and resistance genes.

We present the comparative characterisation of 195 non-aureus staphylococci (NAS) isolates obtained from sheep (n = 125) and humans (n = 70) in Sardinia, Italy, identified at the species level by gap gene polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis with AluI. Isolates were tested phenotypically with a disc diffusion method and genotypically by PCR, for resistance to 11 antimicrobial agents including cationic antiseptic agents. Among the ovine isolates, Staphylococcus epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8) and S. caprae (n = 6) were the most prevalent species, while among human isolates, S. haemolyticus (n = 28) and S. epidermidis (n = 26) were predominant, followed by S. lugdunensis and S. hominis (n = 4). Of the 125 ovine isolates, 79 (63.2%) did not carry any of the resistance genes tested, while the remainder carried resistance genes for at least one antibiotic. The highest resistance rates among ovine isolates were recorded against tetracycline (20.8%), and penicillin (15.2%); none was resistant to methicillin and two exhibited multidrug resistance (MDR); one of which was positive for the antiseptic resistance smr gene. By contrast, most human isolates (59/70, 84.3%) were resistant to ⩾1 antimicrobials, and 41 (58.6%) were MDR. All 52 (74.3%) penicillin-resistant isolates possessed the blaZ gene, and 33 of 70 (47.1%) harboured the mec gene; of these, seven were characterised by the Staphylococcal Chromosomal Cassette (SCCmec) type IV, 6 the type V, 5 of type III and one representative each of type I and type II. The majority (57.1%) was erythromycin-resistant and 17 isolates carried only the efflux msrA gene, 11 the methylase ermC gene and an equal number harboured both of the latter genes. Moreover, 23 (32.8%) were tetracycline-resistant and all but one possessed only the efflux tetK gene. qacA/B and smr genes were detected in 27 (38.6%) and 18 (25.7%) human NAS, respectively. These results underline a marked difference in species distribution and antimicrobial resistance between ovine and human-derived NAS.

The value of the biomarkers cathelicidin, milk amyloid A, and haptoglobin to diagnose and classify clinical and subclinical mastitis.

Timely and objective diagnosis and classification of mastitis is crucial to ensure adequate management and therapeutic decisions. Analyzing specific biomarkers in milk could be advantageous compared with subjective or semiquantitative criteria, such as palpation of the udder in clinical mastitis cases or evaluation of somatic cell count using cow side tests (e.g., California Mastitis Test) in subclinical mastitis quarters. The objective of this study was to investigate the diagnostic value of 3 biomarkers; cathelicidin, milk amyloid A, and haptoglobin for the diagnosis of subclinical and clinical mastitis. Furthermore, the suitability of these biomarkers to differentiate between mild, moderate, and severe clinical mastitis and the influence of different pathogens on biomarker levels was tested. A total of 67 healthy cows, 119 cows with subclinical mastitis, and 212 cows with clinical mastitis were enrolled in the study. Although cathelicidin, haptoglobin, and milk amyloid A were measured in all samples from healthy cows and those with subclinical mastitis, haptoglobin, and cathelicidin results were only available from 121 out of 212 cows with clinical mastitis. Milk amyloid A was measured in all samples. In cows with clinical mastitis, the mastitic quarter and a second healthy quarter serving as a healthy in-cow control quarter were sampled. It was possible to differentiate between healthy quarters, quarters with subclinical mastitis, and quarters with clinical mastitis using all 3 biomarkers. Concerning cathelicidin, thresholds were 0.000 [sensitivity (Se) = 0.83, specificity (Sp) = 0.97] and 0.053 (Se = 0.98, Sp = 0.99) for normalized optical density at 450 nm (NOD450) for differentiating between healthy quarters and quarters with subclinical or clinical mastitis, respectively. Thresholds of 1.28 µg/mL (Se = 0.65, Sp = 0.76) and 1.81 µg/mL (Se = 0.77, Sp = 0.83) for milk amyloid A and 3.65 µg/mL (Se = 0.92, Sp = 0.94) and 5.40 µg/mL mL (Se = 0.96, Sp = 0.99) for haptoglobin were calculated, respectively. Healthy in-cow control quarters from cows with CM showed elevated milk amyloid A and haptoglobin levels compared with healthy quarters from healthy cows. Only the level of milk amyloid A was higher in severe clinical mastitis cases compared with mild ones. In contrast to clinical mastitis, cathelicidin and haptoglobin in subclinical mastitis quarters were significantly influenced by different bacteriological results. The measurement of cathelicidin, milk amyloid A, and haptoglobin in milk proved to be a reliable method to detect quarters with subclinical or clinical mastitis.

The untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci.

This observational study determined the lipidome of cow milk during subclinical intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted approach. Among the pathogens causing bovine IMI, NAS have become the most frequently isolated bacteria from milk samples. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, peptidome, and metabolome, the milk lipidome during mammary gland inflammation remains undisclosed. To cover this gap, we determined the milk lipidome of 17 dairy cows with IMI caused by NAS (NAS-IMI), and we compared the results with those of healthy quarter milk from 11 cows. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,556 measured lipids, the abundance of 597 changed more than 10-fold in quarter milk with NAS-IMI compared with healthy quarters. The results demonstrate the influence of NAS-IMI on the milk lipidome, implying significant changes in lipid species belonging to the family of triacylglycerols and sphingomyelins, and contribute to the understanding of inflammatory processes in the bovine udder, highlighting potential novel biomarkers for improving mastitis diagnostics.

Mycoplasma species isolated from bovine milk collected from US dairy herds between 2016 and 2019.

Determining the species of mycoplasma isolated from culture-positive milk samples is important for understanding the clinical significance of their detection. Between August 2016 and December 2019, 214,518 milk samples from 2,757 dairy herds were submitted to Quality Milk Production Services (QMPS) at Cornell University for mycoplasma culture. From these samples, 3,728 collected from 204 herds were culture positive. Based on the request of herd managers, owners, or veterinarians, 889 isolates from 98 herds were subjected to molecular identification by PCR and amplicon sequencing. The largest proportion of the identified isolates were from New York State (78.1%), while the others came from the eastern United States (17.8%), Texas (2.0%), and New Mexico (2.1%). As expected, Mycoplasma spp. were the most common (855 isolates, 96.2%) and Acholeplasma spp. accounted for the remainder (34 isolates, 3.8%). Mycoplasma bovis was the most prevalent Mycoplasma species (75.1%), followed by M. bovigenitalium (6.5%), M. canadense (5.9%), M. alkalescens (5%), M. arginini (1.7%), M. californicum (0.1%), and M. primatum (0.1%). A portion of the isolates were confirmed as Mycoplasma spp. other than M. bovis but were not identified at the species level (16 isolates, 1.8%) because further information was not requested by the manager, owner, or veterinarian. Mycoplasma bovis was the only species identified in 59 of the 98 herds. However, more than 1 Mycoplasma sp. was identified in 29 herds, suggesting that herd infection with 2 or more mycoplasmas is not uncommon. Moreover, a Mycoplasma sp. other than M. bovis was the only species identified in 8 herds. From the subset of 889 mycoplasma culture-positive isolates from 98 herds, we determined that over a third of the herds had either more than 1 Mycoplasma sp. or a Mycoplasma sp. other than M. bovis detected in their milk samples. In conclusion, we observed that M. bovis is the most common pathogenic Mycoplasma species found in mastitic milk, but other Mycoplasma species are not uncommon. Our results suggest that it is critical to test milk samples for mycoplasmas using diagnostic tests able to identify both the genus and the species.

Molecular mobility changes after high-temperature, short-time pasteurization: An extended time-domain nuclear magnetic resonance screening of ewe milk.

This study presents an extensive investigation on the effect of pasteurization on raw whole ewe milk. Milk samples have been analyzed, throughout lactation (from February to July), by time-domain nuclear magnetic resonance (TD-NMR), collecting the characteristic TD-NMR relaxation parameters, proton longitudinal and transverse relaxation times (1H T1 and T2). Collected data aim at integrating previous NMR works, mainly focusing on dairy model systems (casein and whey proteins solutions and gels, reconstituted skim milk) and cheese, with specific reference to the effect of heat treatments. Whole ewe milk, from a single flock (Sarda sheep breed), was daily analyzed both as untreated (raw) and heat treated with a laboratory-scaled high-temperature, short-time treatment (72°C for 15 and 20 s). Moreover, molecular dynamics in milk were investigated by TD-NMR in different periods of lactation for the first time. As a consequence of high-temperature short-time treatment, 1H T1 and T2 consistently shifted to lower values with respect to raw counterparts. Statistical analysis indicated a significant decrease of T2 in treated samples, to an extent dependent on the heat treatment duration. A subset of dedicated experiments demonstrated that the observed T2 shift is largely ascribable to protein molecular rearrangements and, to a lesser extent, to the interaction of fat globules with proteins or other nonfat components (or both). In light of the crucial importance of detecting the application of a heat treatment to milk, the results reported here suggest TD-NMR relaxation parameters were able to describe heat-induced changes in molecular dynamics and interactions of milk components in a water-rich environment. The use of TD-NMR can be considered a potential suitable technique for quality control and assurance practices in the dairy industry. Upon statistical validation of methods, the application of TD-NMR in the dairy industry would take advantage of its low cost, reliability, and robustness.

A gas chromatography-mass spectrometry untargeted metabolomics approach to discriminate Fiore Sardo cheese produced from raw or thermized ovine milk.

Thermization is a sub-pasteurization heat treatment of cheese milk (at 57-68°C for 15-30 s) aimed to reduce the number of undesirable microbial contaminants with reduced heat damage to the indigenous milk enzymes. In this work, the effects of milk thermization on the compositional parameters, proteolysis indices, free fatty acid levels, and low molecular weight metabolite profiles of ovine cheese were studied. Cheese samples at different ripening stages and produced in 2 different periods of the year were analyzed. While the effects of milk thermization on cheese macro-compositional parameters and free fatty acid levels were not evident due to the predominant effects of milk seasonality and cheese ripening stage, the gas chromatography-mass spectrometry based metabolomics approach of ovine cheese produced from raw and thermized milk highlighted strong differences at the metabolite level. Discriminant analysis applied to gas chromatography-mass spectrometry data provided an excellent classification model where cheese samples were correctly classified as produced from raw or thermized milk. The metabolites that mostly changed due to the thermization process belonged to the classes of free amino acids and saccharides. Gas chromatography-mass spectrometry-based metabolomics has proven to be a valid tool to study the effect of mild heat treatments on the polar metabolite profile in ovine cheese.

Liver proteomics of gilthead sea bream (Sparus aurata) exposed to cold stress.

The gilthead sea bream (Sparus aurata, L.) is very sensitive to low temperatures, which induce fasting and reduced growth performances. There is a strong interest in understanding the impact of cold on fish metabolism to foster the development and optimization of specific aquaculture practices for the winter period. In this study, an 8 week feeding trial was carried out on gilthead sea bream juveniles reared in a Recirculated Aquaculture System (RAS) by applying a temperature ramp in two phases of four weeks each: a cooling phase from 18 °C to 11 °C and a cold maintenance phase at 11 °C. Liver protein profiles were evaluated with a shotgun proteomics workflow based on filter-aided sample preparation (FASP) and liquid chromatography-mass spectrometry (LC-ESI-Q-TOF MS/MS) followed by label-free differential analysis. Along the whole trial, sea breams underwent several changes in liver protein abundance. These occurred mostly during the cooling phase when catabolic processes were mainly observed, including protein and lipid degradation, together with a reduction in protein synthesis and amino acid metabolism. A decrease in protein mediators of oxidative stress protection was also seen. Liver protein profiles changed less during cold maintenance, but pathways such as the methionine cycle and sugar metabolism were significantly affected. These results provide novel insights on the dynamics and extent of the metabolic shift occurring in sea bream liver with decreasing water temperature, supporting future studies on temperature-adapted feed formulations. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011059.

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis.

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.

Evaluation of milk cathelicidin for detection of dairy sheep mastitis.

Mastitis due to intramammary infections is one of the most detrimental diseases in dairy sheep farming, representing a major cause of reduced milk productions and quality losses. In particular, subclinical mastitis presents significant detection and control problems, and the availability of tools enabling its timely, sensitive, and specific detection is therefore crucial. We have previously demonstrated that cathelicidins, small proteins implicated in the innate immune defense of the host, are specifically released in milk of mastitic animals by both epithelial cells and neutrophils. Here, we describe the development of an ELISA for milk cathelicidin and assess its value against somatic cell counts (SCC) and bacteriological culture for detection of ewe mastitis. Evaluation of the cathelicidin ELISA was carried out on 705 half-udder milk samples from 3 sheep flocks enrolled in a project for improvement of mammary health. Cathelicidin was detected in 35.3% of milk samples (249/705), and its amount increased with rising SCC values. The cathelicidin-negative (n=456) and cathelicidin-positive (n=249) sample groups showed a clear separation in relation to SCC, with median values of 149,500 and 3,300,000 cells/mL, respectively. Upon bacteriological culture, 20.6% (145/705) of the milk samples showed microbial growth, with coagulase-negative staphylococci being by far the most frequent finding. A significant proportion of all bacteriologically positive milk samples were positive for cathelicidin (110/145, 75.9%). Given the lack of a reliable gold standard for defining the true disease status, sensitivity (Se) and specificity (Sp) of the cathelicidin ELISA were assessed by latent class analysis against 2 SCC thresholds and against bacteriological culture results. At an SCC threshold of 500,000 cells/mL, Se and Sp were 92.3 and 92.3% for cathelicidin ELISA, 89.0 and 94.9% for SCC, and 39.4 and 93.6% for bacteriological culture, respectively. At an SCC threshold of 1,000,000 cells/mL, Se and Sp were 93.3 and 91.9% for cathelicidin ELISA, 80.0 and 97.1% for SCC, and 39.4 and 93.5% for bacteriology, respectively. In view of the results obtained in this study, the measurement of cathelicidin in milk by ELISA can provide added Se while maintaining a high Sp and may therefore improve detection of subclinical mastitis.

Evaluation of milk cathelicidin for detection of bovine mastitis.

Mastitis due to intramammary infection is one of the most economically relevant diseases in dairy cows, causing reductions in milk quality and quantity. Currently, mastitis monitoring is based on somatic cell count (SCC) and bacteriologic culture (BC) of milk. Nevertheless, inflammation-specific protein markers might provide more sensitive and reliable assays, enabling immunoassay-based screening strategies. Cathelicidin is an inflammatory protein released in milk that has recently demonstrated fair reliability and diagnostic potential for ewe mastitis. To assess its performance in cows, 531 quarter milk samples from 2 herds were tested using cathelicidin ELISA, SCC, and BC. We found that 29.0% of samples were positive for cathelicidin, 18.8% had SCC >200,000 cells/mL, and 13.7% were BC-positive. Cathelicidin showed a strong positive correlation with SCC as demonstrated by receiver operating characteristics curve analysis and by the clustering of cathelicidin-negative and cathelicidin-positive samples in association with low and high SCC values, respectively. For evaluating the diagnostic performance of a novel test, BC cannot be considered a reliable gold standard for true disease status because of its known limitations. Therefore, we assessed the sensitivity (Se) and specificity (Sp) of the milk cathelicidin ELISA using a latent class analysis approach together with BC and SCC by considering different diagnostic thresholds to identify the preferred Se/Sp combination. We modeled conditional dependence of cathelicidin and SCC to account for their close association. The cathelicidin ELISA showed higher Se than SCC and BC for almost all threshold combinations. In fact, at the best-performing threshold combination, the Se of cathelicidin was 80.6%, 6.2 percentage points higher than that of SCC >200,000 cells/mL (74.4%) and similar to that of SCC >100,000 cells/mL (80.2%). Most importantly, this Se was obtained with a loss in Sp of only 1.4 percentage points compared with SCC >200,000 cells/mL (94.9% Sp for cathelicidin vs. 96.3% for SCC >200,000). The limited Se of BC (38.8%) was also confirmed in this study, and BC showed a slightly lower Sp than both cathelicidin and SCC for most of threshold combinations. This study confirmed that cathelicidin is released in the milk of cows with mastitis and that its presence is highly correlated with SCC. The measurement of cathelicidin by ELISA may hold significant potential for improving the sensitivity of mastitis detection in dairy cows while maintaining high specificity.

Quality and fatty acid profile of the milk of indigenous goats subjected to different local diets in Tunisian arid lands.

The study tested the hypothesis that certain pastoral forages and olive by-products, available in arid areas, may positively influence fatty acid composition and physicochemical properties of goat's milk. Thirty indigenous goats (body weight = 25.2 kg; age = 4.1 years) were allocated to three groups. During 60 days, the goats received ad libitum either dried olive leaves + Stipa tenacissima (group OL), khortane grass hay (group Ko) or oat hay (control diet, group OH). Milk samples were collected and analysed for total solids, fat, protein, lactose and ash content and fatty acid profile. Average milk yield did not statistically differ among groups. Milk total solids from OL group were higher in comparison with Ko and C groups (15.3, 14.7 and 14.5%, respectively; p < 0.05). Fat content was also higher for the OL group as compared to the other groups (5.44 vs. 5.01 and 4.66%, respectively, for Ko and OH). No significant differences were observed for the milk content of lactose, protein and ash. The percentage of saturated fatty acids of total milk fat was higher in OL and Ko groups compared to the C group (p < 0.001); the milk whereof was characterized by the highest percentage of monounsaturated (p < 0.01) and total unsaturated fatty acids. Milk fat of Ko and C groups showed significantly higher proportions of rumenic (CLA cis-9 trans-11) and vaccenic acids (C18:1 trans-11) compared to OL milk. The feeding system based on Stipa tenacissima and dried olive leaves resulted in the milk lowest proportion of trans-fatty acids and the highest proportion of polyunsaturated ω3-fatty acids (p < 0.05).

Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22% of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer.

Effect of whey concentration on protein recovery in fresh ovine ricotta cheese.

Ricotta cheese, particularly the ovine type, is a typical Italian dairy product obtained by heat-coagulation of the proteins in whey. The aim of this work was to investigate the influence of whey protein concentration, obtained by ultrafiltration, on yield of fresh ovine ricotta cheese. Ricotta cheeses were obtained by thermocoagulation of mixtures with protein content of 1.56, 3.10, 4.16, and 7.09g/100g from the mixing of skim whey and ultrafiltered skim whey. A fat-to-protein ratio of 1.1 (wt/wt) was obtained for all mixtures by adding fresh cream. The initial mixtures, as well as the final ricotta cheeses, were analyzed for their composition and by SDS-PAGE. Protein bands were quantified by QuantityOne software (Bio-Rad, Hercules, CA) and identified by liquid chromatography-tandem mass spectrometry. Significant differences in the composition of the ricotta cheese were observed depending on protein concentration. Particularly, ricotta cheese resulting from the mixture containing 7.09g/100g of protein presented higher moisture (72.88±1.50g/100g) and protein (10.18±0.45g/100g) contents than that prepared from the mixture with 1.56g/100g of protein (69.52±1.75 and 6.70±0.85g/100g, respectively), and fat content was lower in this sample (12.20±1.60g/100g) compared with the other treatments, with mean values between 15.72 and 20.50g/100g. Each protein fraction presented a different behavior during thermocoagulation. In particular, the recovery of ß-lactoglobulin and α-lactalbumin in the cheese increased as their content increased in the mixtures. It was concluded that concentrating ovine rennet whey improved the extent of heat-induced protein aggregation during the thermal coagulation process. This resulted in a better recovery of each protein fraction in the product, and in a consequent increase of ricotta cheese yield.

Genomic Characterization of Mycoplasma arginini Isolated from a Housefly on a Dairy Farm and Comparison with Isolates from Bovine Milk and Lung Tissue.

Mycoplasma mastitis can be highly contagious, unresponsive to treatment, and cause severe economic problems in affected herds. Notable routes of Mycoplasma spp. transmissions are contaminated milking equipment and animal contact through respiratory secretions. Only a few studies report the environment as a possible source of infection. Our group studied the presence of pathogens in houseflies (Musca domestica) in a New York State dairy in the United States. Among others, a Mycoplasma spp. was found in the gut of a housefly captured in the sick pen and identified as M. arginini. Here, we characterized its genome and investigated its relatedness with eight isolates from milk, one isolate from lung tissue collected in the same dairy, and five other dairies in New York State. We applied whole-genome sequencing and phylogenetic analysis based on the sequences of the 16S rRNA gene and 76 conserved proteins. We also assessed an in silico virulence profile by considering a panel of 94 putative virulence genes. As a result of the genome analysis, the housefly M. arginini isolate was highly similar to the milk isolates; interestingly, the similarity was highest with M. arginini isolated from milk on the same dairy farm where the housefly was captured. The housefly and milk M. arginini isolates possessed 54 of the 94 pathogenicity genes considered. Our data support the hypothesis that houseflies are carriers of Mycoplasma spp. and can be considered within the possible roots of environmental transmission of infection in dairy cows. Nevertheless, M. arginini pathogenicity will need to be investigated with dedicated studies. IMPORTANCE It is critical to control the spread of bovine mastitis caused by Mycoplasma spp., as this disease can be highly contagious and have a severe economic impact on affected dairies. A better understanding of possible transmission routes is crucial for infection control and prevention. Based on our data, the composite milk isolates are genetically similar to the housefly isolate. This provides evidence that the same Mycoplasma species found in milk and associated with mastitis can also be isolated from houseflies captured in the dairy environment.

[Pilot study for the assessment of perceived critical organizational aspects in a hospital of Local Health Authority of Cagliari, Italy]. / Studio pilota per la valutazione delle criticità organizzative percepite dai lavoratori presso un presidio ospedaliero della ASL 8 di Cagliari.

INTRODUCTION: In Italy, the effectiveness of public health services must be assessed trhoug the assessment of efficency of employees. OBJECTIVE: To acquire from this activity useful information to assess and promote the well being at work. METHODS: During the assessment activities, a questionnaire was administered to assess the efficency of the management in manage perceived critical organizational aspects. RESULTS: The 44-58% consider not sufficient the management efficency in manage perceived critical organizational aspects, exspecially for quality of services, communication, vocational training and risks for worker's health and safety. CONCLUSIONS: The study provide useful suggestions to better assess and manage the risk of work-related stress and exploit the key consultant role of occupational physician in the organizational context.

[Assessment of congenital malformation risk in the progeny of the military and civilian personnel of the Salto di Quirra military base: preliminary results]. / Valutazione del rischio di malformazioni congenite nella progenie del personale militare e civile del Poligono Interforze del salto di Quirra: risultati preliminari.

INTRODUCTION: We evaluated the congenital malformation rate in the progeny of the personnel of the Salto di Quirra military base in Sardinia. METHODS: During 2011, we gathered questionnaire information on the reproductive history of 389 employees, more then 99% of those eligible for routine health surveillance. RESULTS: the observed congenital malformation rate (20.1 x 10(-3), 95% CI 6.3 - 33.8) was lower than that reported by the Italian Registries of Congenital Malformations, and it did not vary by exposure to radiofrequency, elf electromagnetic fields, and solvents, and by jobs associated with alleged exposure to nanoparticles or alpha radiation. CONCLUSIONS: Our findings suggest that the documented or alleged occupational exposures among the PISQ workforce did not increase the congenital malformation rate in the progeny.

Comparative secretome analysis of Staphylococcus aureus strains with different within-herd intramammary infection prevalence.

Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of ß-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

A Paradox in Bacterial Pathogenesis: Activation of the Local Macrophage Inflammasome Is Required for Virulence of Streptococcus uberis.

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host-pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140 J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1ß from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1ß was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.