The secretome of Staphylococcus aureus strains with opposite within-herd epidemiological behavior affects bovine mononuclear cell response.

Staphylococcus aureus modulates the host immune response directly by interacting with the immune cells or indirectly by secreting molecules (secretome). Relevant differences in virulence mechanisms have been reported for the secretome produced by different S. aureus strains. The present study investigated the S. aureus secretome impact on peripheral bovine mononuclear cells (PBMCs) by comparing two S. aureus strains with opposite epidemiological behavior, the genotype B (GTB)/sequence type (ST) 8, associated with a high within-herd prevalence, and GTS/ST398, associated with a low within-herd prevalence. PBMCs were incubated with different concentrations (0%, 0.5%, 1%, and 2.5%) of GTB/ST8 and GTS/ST398 secretome for 18 and 48 h, and the viability was assessed. The mRNA levels of pro- (IL1-ß and STAT1) and anti-inflammatory (IL-10, STAT6, and TGF-ß) genes, and the amount of pro- (miR-155-5p and miR-125b-5p) and anti-inflammatory (miR-146a and miR-145) miRNAs were quantified by RT-qPCR. Results showed that incubation with 2.5% of GTB/ST8 secretome increased the viability of cells. In contrast, incubation with the GTS/ST398 secretome strongly decreased cell viability, preventing any further assays. The GTB/ST8 secretome promoted PBMC polarization towards the pro-inflammatory phenotype inducing the overexpression of IL1-ß, STAT1 and miR-155-5p, while the expression of genes involved in the anti-inflammatory response was not affected. In conclusion, the challenge of PBMC to the GTS/ST398 secretome strongly impaired cell viability, while exposure to the GTB/ST8 secretome increased cell viability and enhanced a pro-inflammatory response, further highlighting the different effects exerted on host cells by S. aureus strains with epidemiologically divergent behaviors.

Chronic intramammary infection by Listeria monocytogenes in a clinically healthy goat - a case report.

BACKGROUND: Listeria monocytogenes is a ubiquitous Gram-positive bacterium responsible for a severe foodborne disease in humans, and contaminated dairy products can be an important source of infection. Typically, infected dairy ruminants show clinical manifestations including encephalitis, septicemia, abortion, and diarrhea, but may also become asymptomatic carriers and shed L. monocytogenes in the feces acting as an important source of viable bacteria. Isolation from individual goat milk has been documented very rarely, and chronic, asymptomatic intramammary infection by L. monocytogenes with continuous milk shedding of viable bacteria has never been described in this dairy species. CASE PRESENTATION: At the routine controls, cheese and bulk milk were positive for L. monocytogenes in a herd of 200 lactating Alpine goats, but none showed clinical signs of listeriosis. Individual milk was subjected to bacterial culture and a clinically healthy goat was identified as affected by a chronic intramammary infection (IMI) by L. monocytogenes. The goat had never shown clinical signs of mastitis or other diseases. Her right half-udder milk was positive to L. monocytogenes in two consecutive samples collected one week apart, as demonstrated by bacterial culture and molecular analysis. Mammary tissues collected after culling were also positive to L. monocytogenes by culture. Histological examination highlighted a chronic interstitial mastitis with leukocyte infiltration, atrophy of the alveoli and presence of corpora amylacea. Immunohistochemistry (IHC) and immunofluorescence (IF) confirmed the presence of high numbers of bacteria in the lumen of mammary alveoli, with intracellular bacteria mainly located in macrophages, but also present in neutrophils and epithelial cells. After culling of the positive goat, bulk tank milk tested negative to L. monocytogenes at the following controls. CONCLUSION: This study demonstrates that L. monocytogenes can establish a chronic, subclinical IMI in goats with high numbers of bacteria shed in milk, representing a source of contamination for the herd and its dairy products. This underscores the importance of frequently monitoring all dairy herds that sell directly milk and/or fresh cheese and indicates that a chronic L. monocytogenes IMI should also be considered as source of bacteria when bulk tank milk contamination is detected in a dairy goat farm.

Effects of herd and physiological status on variation of 16 immunological and inflammatory parameters in dairy cows during drying off and the transition period.

During drying off and transition period, cows are subject to changes in endocrine status, metabolic stressors and altered immune functions, which could lead to an increased risk of disease. To expand our knowledge on the immune/inflammatory status and to identify markers to define cow status during this interval, the pattern of 9 different cellular parameters, 5 cytokines, 2 enzymes and 3 cellular ratios in blood samples were assessed in 15 primiparous cows belonging to three different dairy herds in Lombardy. Our data showed that the variation of almost all parameters was influenced by the physiological period in which the samples were collected, except for apoptosis, IL-1ß, IL-6, lysozyme and granulocyte/monocyte ratio. Several markers were directly correlated either to the herd alone (IL-1ß, IL-6, lysozyme, granulocyte/lymphocyte ratio and granulocyte/monocyte ratio) or in association with the sampling time (white blood cell count, necrosis, lymphocytes count, CD4+ lymphocytes proportion). Hierarchical cluster analysis identified three herd-associated sample clusters showing different frequency along the follow-up period. The results of this field study highlight the importance of the herd factor in the immune/inflammatory response. Furthermore, these results suggest that cellular parameters are probably the most suitable markers to define cow status during drying-off and the peripartum period.

Molecular characteristics of bap-positive Staphylococcus aureus strains from dairy cow mastitis.

The biofilm-associated protein (Bap) of Staphylococcus aureus is a high molecular weight cell-wall-anchored protein involved in biofilm formation, first described in bovine mastitis strains from Spain. So far, studies regarding Bap were mainly based on the Spanish strain V329 and its mutants, but no information on the genetic variability of bap-positive Staph. aureus strains is yet available in the literature. The present study investigated the molecular characteristics of 8 bap-positive Staph. aureus strains from subclinical bovine mastitis, isolated in 5 herds; somatic cell counts (SCC) of milk samples were also registered. Strains were characterised using MLST, SPA typing and microarray and the results were compared with V329. All isolates from this study and V329 were assigned to ST126, t605, but some molecular differences were observed. Only herd A and B strains harboured the genes for ß-lactams resistance; the leukocidin D/E gene, a type I site-specific deoxyribonuclease subunit, 3rd locus gene and serin-protease A and B were carried by all strains, but not by V329, while serin-protease E was absent in V329 and in another isolate. Four isolates and V329 harboured the fibronectin-binding protein B gene. SCC showed the highest value in the milk sample affected by the only strain carrying all the virulence factors considered. Potential large variability of virulence was evidenced among V329 and all bap-positive Staph. aureus strains considered: the carriage of fnb could enhance the accumulation of biofilm, but the lack of lukD/E and splA, B or E might decrease the invasiveness of strain.

Non-aureus staphylococci and mammaliicocci isolated from bovine milk in Italian dairy farms: a retrospective investigation.

Non-aureus staphylococci and mammaliicocci (NASM) are associated with bovine mastitis and increased milk somatic cell count (SCC) but their relationships with mammary gland health at the species level are not clearly defined. Regional differences have also been reported in their specific prevalence. The implementation of MALDI-TOF MS in milk microbiology is generating large and dependable datasets with the potential of providing useful epidemiological information. We present the retrospective analysis of 17,213 milk samples sent to our laboratory in 2021-2022, including 13,146 quarter samples from cows with subclinical (SCM) or clinical mastitis (CM) from 104 farms, and 4,067 composite herd survey (HS) samples from 21 farms. NASM were isolated from 21.12% of SCM, 11.49% of CM, and 15.59% of HS milk samples. The three most frequently identified NASM in SCM milk were Staphylococcus chromogenes (33.33%), S. haemolyticus (26.07%), and S. epidermidis (10.65%); together with S. microti and S. hyicus, these species were significantly more prevalent in quarters with SCM (p < 0.05). The three most frequently identified NASM in CM milk were S. chromogenes (31.69%), S. haemolyticus (21.42%), and Mammaliicoccus sciuri (18.38%), although no significant associations were found between these NASM species and CM. The three most frequently identified NASM in HS milk were S. chromogenes (44.49%), S. epidermidis (17.84%), and S. haemolyticus (17.23%), with S. chromogenes being isolated in all the farms sending HS milk (100%). In conclusion, this retrospective study provides the first information on the NASM species isolated from cow milk in Italy, expanding our knowledge on the epidemiology of NASM at the species level and providing further insights into their relationships with mammary gland health in modern dairy farms.

An update on non-aureus staphylococci and mammaliicocci in cow milk: unveiling the presence of Staphylococcus borealis and Staphylococcus rostri by MALDI-TOF MS.

Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM.

Multidrug-Resistant Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in a Dairy Herd: Distribution and Antimicrobial Resistance Profiles.

This study investigated the presence, distribution, and antimicrobial resistance profiles of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in a dairy herd located in Northern Italy. The feces of clinically healthy calves, their mothers, and the cows treated for mastitis, as well as water, environmental samples, and waste milk were collected and subjected to bacteriological culture on CHROMagarTM ESBL plates. A questionnaire was administered to identify risk factors. The isolates were identified as E. coli by MALDI-TOF MS and subjected to the double-disk synergy test (DDST) and minimal inhibitory concentration (MIC) assay. As a result, ESBL E. coli was isolated from the feces of 28 of 37 (75.67%) calves, the feces of 2 of 3 (66.67%) treated cows, 8 of 14 (57.15%) environmental samples, and waste milk. All ESBL isolates showed multiple resistances and were categorized as multidrug-resistant (MDR). Several risk factors for ESBL E. coli selection and diffusion were identified, including lack of routine cleaning of calf feeding and housing equipment, administration of waste milk to male calves, and blanket dry cow therapy. In conclusion, this study highlighted the presence of MDR, ESBL E. coli in the feces of most dairy calves, and their association with different sample sources. Accordingly, adding to the prudent use of antibiotics, the adoption of adequate farm hygiene and biosecurity measures might also help prevent the spread and transmission of ESBL E. coli within the herd.

Pathogen group-specific risk factors for intramammary infection in water buffalo.

A cross-sectional study was conducted to estimate the prevalence of intramammary infection (IMI) associated bacteria and to identify risk factors for pathogen group-specific IMI in water buffalo in Bangladesh. A California Mastitis Test (CMT) and bacteriological cultures were performed on 1,374 quarter milk samples collected from 763 water buffalo from 244 buffalo farms in nine districts in Bangladesh. Quarter, buffalo, and farm-related data were obtained through questionnaires and visual observations. A total of 618 quarter samples were found to be culture positive. Non-aureus staphylococci were the predominant IMI-associated bacterial species, and Staphylococcus (S.) chromogenes, S. hyicus, and S. epidermidis were the most common bacteria found. The proportion of non-aureus staphylococci or Mammaliicoccus sciuri (NASM), S. aureus, and other bacterial species identified in the buffalo quarter samples varied between buffalo farms. Therefore, different management practices, buffalo breeding factors, and nutrition were considered and further analyzed when estimating the IMI odds ratio (OR). The odds of IMI by any pathogen (OR: 1.8) or by NASM (OR: 2.2) was high in buffalo herds with poor milking hygiene. Poor cleanliness of the hind quarters had a high odds of IMI caused by any pathogen (OR: 2.0) or NASM (OR: 1.9). Twice daily milking (OR: 3.1) and farms with buffalo purchased from another herd (OR: 2.0) were associated with IMI by any pathogen. Asymmetrical udders were associated with IMI-caused by any bacteria (OR: 1.7). A poor body condition score showed higher odds of IMI by any pathogen (OR: 1.4) or by NASM (OR: 1.7). This study shows that the prevalence of IMI in water buffalo was high and varied between farms. In accordance with the literature, our data highlight that IMI can be partly controlled through better farm management, primarily by improving hygiene, milking management, breeding, and nutrition.

Different Immune Control of Gram-Positive and Gram-Negative Mammary Infections in Dairy Cows.

In the dairy industry, bovine mastitis represents a major concern due to substantial production losses and costs related to therapies and early culling. The mechanisms of susceptibility and effective response to intra-mammary infections are still poorly understood. Therefore, we investigated innate immunity in acellular bovine skim milk through cytofluorimetric analyses of bacterial killing activity against both Gram-positive and Gram-negative pathogens. Freshly cultured E. coli and S. aureus strains were incubated with colostrum and milk samples at different lactation time points from two groups of cows, purportedly representing mastitis-resistant and mastitis-susceptible breeds; bacterial cells were analyzed for vitality by flow cytometry following incorporation of vital dyes. N-acetyl-ß-D-glucosaminidase (NAGase) activity was also investigated in milk and colostrum samples. Our findings revealed that colostrum and milk bacterial killing activity was greater against S. aureus compared to E. coli., with this activity correlated with milk NAGase levels. Furthermore, both killing of S. aureus and NAGase activity were negatively correlated to the elapsed time of lactation. Interestingly, samples from the allegedly mastitis-resistant breed displayed higher bacterial killing and NAGase activities. Our study suggests that diverse control mechanisms are exerted against Gram-positive and Gram-negative pathogens in the mammary glands of cows, probably beyond those already described in the literature.

Duplex real-time PCR assay for rapid identification of Staphylococcus aureus isolates from dairy cow milk.

Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase-or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or-negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detect Staph. aureus mammary infections avoiding bacteriological analysis.

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs.

Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis.

Staphylococcus aureus is one of the most important pathogens associated with bovine mastitis. Recent studies have shown that Staph. aureus strains may differ in virulence, and in their ability to disseminate across commercial dairy herds. The goal of this study was to determine whether Staph. aureus isolates differed in their ability to colonize mammary tissue, and whether such differences could be related to molecular characteristics. Quarter milk and mammary tissues of 22 cows from two dairy herds, were collected at slaughter and bacteriological analysis was performed. All Staph. aureus isolates were characterized by Pulsed Field Gel Electrophoresis (PFGE) and microarray. Overall 45 mammary quarters were infected and 20 Staph. aureus isolates were identified. The bacteria were mostly recovered from both milk and tissue of the same quarter in significantly higher numbers from herd A cows compared with herd B. Molecular characterization of the isolates showed distinct PFGE profiles for isolates from each herd. Differences in virulence factors between herds A and B isolates were evidenced The genes for enterotoxin D, J and R were present in herd A, those for G, I, N, M, O and U were shown in herd B, whilst both components of the leukocidin lukD/E genes were only carried by herd A isolates. Furthermore, all herd A isolates showed ß-haemolysin activity, which was absent in all but one isolate from herd B. Therefore our data indicate that Staph. aureus isolates showing differences in their ability to disseminate and colonize across quarters, also have significantly different virulence characteristics.

Molecular Tools to Identify and Characterize Malignant Catarrhal Fever Viruses (MCFV) of Ruminants and Captive Artiodactyla.

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

Survey of Staphylococcus aureus carriage by free-living red deer (Cervus elaphus): Evidence of human and domestic animal lineages.

Staphylococcus aureus is a pathogen that can affect multiple host species. Evidence of transmission between humans and animals and among different animal species has been reported in recent years. In this study, we investigated 284 free-living red deer (Cervus elaphus) in the Central Italian Alps to assess the prevalence and molecular characteristics of S. aureus in nasal and intestinal samples in relation to host features and environmental factors. A prevalence of 90%, 26.2% and 10.7% of S. aureus was detected in nasal rectal swabs and faeces, respectively. Calves had a higher probability of being S. aureus intestinal carriers than adults, especially in females when considering faecal samples. Clonal complex (CC) 425 was the most prevalent lineage (61.5%). This is a lineage known to be widespread in both domestic and free-living animals. It was followed by CC2671 (15.4%) and CC350 (6.4%). A high rate of the phage-borne virulence factor lukM/lukF-P83 was detected in CC425 and CC350. Further lineages, which are known to occur in both humans and animals, were detected sporadically in red deer faeces only, that is, CC7, CC9, CC121 and CC707, harbouring the genes of the penicillinase operon and a gene for macrolide resistance (CC9 and CC121). Methicillin resistance genes mecA and mecC were not found. Our results suggest that free-living red deer may be reservoir for S. aureus in Alpine habitats.

Changes in the lipidome of water buffalo milk during intramammary infection by non-aureus Staphylococci.

This study aimed to determine the lipidome of water buffalo milk with intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted lipidomic approach. Non-aureus Staphylococci are the most frequently isolated pathogens from dairy water buffalo milk during mastitis. A total of 17 milk samples from quarters affected by NAS-IMI were collected, and the lipidome was determined by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results were compared with the lipidome determined on samples collected from 16 healthy quarters. The study identified 1934 different lipids, which were classified into 15 classes. The abundance of 72 lipids changed in NAS-IMI milk compared to healthy quarters. Significant changes occurred primarily in the class of free fatty acids. The results of this study provided first-time insight into the lipidome of dairy water buffalo milk. Moreover, the present findings provide evidence that NAS-IMI induces changes in water buffalo milk's lipidome.

Peptidomic changes in the milk of water buffaloes (Bubalus bubalis) with intramammary infection by non-aureus staphylococci.

Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

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.

Effect of Weeping Teats on Intramammary Infection and Somatic Cell Score in Dairy Goats.

Mastitis is the most common disease affecting dairy goats and causing economic losses. Although it is accepted that increased somatic cell count (SCC) is mainly a response to infection, its reliability for subclinical mastitis detection in goats is controversial. Indeed, many physiological and extrinsic variables can increase SCC, including breed, parity, age, stage of lactation, seasonal variations, and milking methods. In some animals, milk-secreting tissue is present in the wall of the teat and, in some instances, milk can filter through pores in the skin to the udder surface. This condition is known as "weeping teat" (WT). In these animals, mammary tissue might be prone to develop bacterial infections, although limited information is provided. Weeping teat seems to have a genetic background and is reported to be especially found in goat breeds selected for high milk production. Moreover, it is observed a genetic correlation between WT and decreased milk yield as well as increased somatic cell scores (SCS). Since information on this topic is very limited, this study aimed at investigating any possible relationship between WT, high SCC, and the presence of bacteria in goat milk. Alpine goat farms in Northern Italy were selected based on the presence of WT. Each herd was divided into two age-matched groups, identified as case (WT+) and control (WT-). Half-udder milk samples were collected aseptically at three timepoints; bacteriological analysis was performed, and SCC were determined and transformed in SCS. There was a positive association between SCS and the presence of bacteria in milk (P = 0.037) overall, whereas WT udder defect was associated with positive bacterial culture in just one herd (P = 0.053). Thus, this herd was further investigated, repeating the sampling and the analysis on the following year. The positive association between high SCS and the presence of bacteria in milk was then confirmed (P = 0.007), whereas no association with WT condition was found. These results indicate that WT defect is usually unrelated to both the outcome of milk bacterial culture and SCS. As a side outcome, we could confirm the role of bacterial infection in increasing SCS.

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.

Broad-spectrum activity against bacterial mastitis pathogens and activation of mammary epithelial cells support a protective role of neutrophil cathelicidins in bovine mastitis.

Cathelicidins are peptide components of the innate immune system of mammals. Apart from exerting a direct antibiotic activity, they can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied, but there is a lack of published information on their expression and activities in the context of mastitis. The aims of this study were to investigate the expression of the bovine cathelicidins BMAP-27, BMAP-28, Bac5, and indolicidin in healthy and infected mammary tissue and in lipopolysaccharide (LPS)-treated cells, to determine their activities against bacteria isolated from bovine mastitis, and to examine their potentials to trigger defense responses in bovine mammary cells. The genes were found to be upregulated in LPS-stimulated neutrophils, but not in infected quarters or epithelial cells. All peptides showed a variably broad spectrum of activity against 28 bacterial isolates from bovine mastitis (MIC values, 0.5 to 32 microM), some of which were antibiotic resistant. The activity of each peptide was significantly enhanced when it was pairwise tested with the other peptides, reaching the synergy threshold when indolicidin was present. The bactericidal activity was sensitive to milk components; BMAP-27 and -28 were highly effective in mastitic bovine milk and inhibited in milk from healthy cows. Both peptides were also active in whey and in blood serum and triggered the expression of tumor necrosis factor alpha (TNF-alpha) in bovine mammary epithelial cells. Our results indicate multiple roles for the bovine cathelicidins in mastitis, with complementary and mutually enhanced antimicrobial activities against causative pathogens and the capacity to activate host cells.