Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

Presence of pathogen DNA in milk harvested from quarters is associated to changes in cows' milk yield and composition.

BACKGROUND: Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows' udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available. RESULTS: Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens. CONCLUSIONS: The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable - especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment.

Responsible antibiotic use labeling and consumers' willingness to buy and pay for fluid milk.

Concerns about antibiotic resistant infections in the United States have called for reduction of antibiotic use in livestock, including dairy cattle. Although effective in curbing antibiotic use, universal organic dairy farming would be impractical and unattainable due to its high land and premium demands. The US Department of Agriculture's organic certification, which completely eliminates antibiotic use in milk production, also raises animal welfare concerns, as it could discourage the use of antibiotics even to treat indicated diseases. Therefore, a proposed alternative for US consumers is a label indicating the responsible antibiotic use (RAU) - not complete elimination - that would minimize antibiotics more than conventional (unlabeled) milk and maximize animal welfare more than organic milk. Our goal was to determine consumers' (1) self-reported preference and (2) willingness to pay for this hypothetical RAU label of milk relative to existing substitutes in organic and unlabeled fluid milk. We conducted (1) a nationally representative survey of US adults and (2) a randomized non-hypothetical experimental Becker-Degroot-Marschak auction with real money and real milk. Although almost half of the survey participants (48.5%) responded that they would buy a RAU-labeled milk, consumers in the experimental auction refused to pay a significant premium for the milk compared with unlabeled milk (mean willingness to pay (95% confidence interval) per half-gallon: $1.92 ($1.65-$2.19) for RAU-labeled milk versus $1.86 ($1.58-$2.13) for unlabeled milk). These results suggest that consumers' survey-identified preferences for RAU-labeled milk could reflect either social desirability bias or a genuine preference for which, however, consumers simply will not pay a significant premium. The study provides preliminary data for future exploration of marketability of the proposed RAU label in the United States and demonstrates the benefits of using complementary survey and experimental auction approaches to understand the potential market for a new dairy product.

Technical note: Development of multiplex PCR assays for the molecular characterization of Streptococcus uberis strains isolated from bovine mastitis.

Streptococcus uberis is an important causative agent for clinical and subclinical mastitis in dairy cattle. The aim of this study was to develop 2 multiplex PCR assays (mPCR) for the simultaneous detection of virulence factors and housekeeping genes for use when investigating the genetic variability and distribution of Strep. uberis virulence factors. The tuf, cpn60, pauA, sodA, sua, oppF, and gapC genes were grouped in assay 1 (mPCR1) and the hasA, hasB, and hasC genes were included in assay 2 (mPCR2). The detection limits were 11.8 pg and 5.9 pg of DNA for mPCR1 and mPCR2, respectively. The 2 mPCR assays were validated with 56 Strep. uberis strains isolated from mastitis milk samples collected from different bovine herds in northern Italy. Results revealed that gapC and oppF were detected in 98.2% of the strains, whereas sua and hasC genes were detected in 94.6 and 89.2% of the strains, respectively. The most common pattern was gapC+, oppF+, cpn60+, sua+, sodA+, pauA+, tuf+, hasA+, hasB+, and hasC+, which appeared in 59% of the strains analyzed. The molecular assays developed in the present study represent a powerful tool for the evaluation of virulence pattern distribution in Strep. uberis strains associated with intramammary infections.

Sand bedding as a reservoir for Lactococcus garvieae dissemination in dairy farms.

Lactococcus garvieae is now recognized as a species with clinical significance for human and veterinary medicine. The aim of this study was to evaluate the presence of this pathogen in sand bedding and milk samples. Two farms in Minnesota with problems of clinical and subclinical mastitis due to streptococci-like organisms were selected. Twenty-four Lactococcus garvieae isolates from sand bedding and 18 isolates from quarter milk were comparatively studied using a genotypic approach. RAPD (random amplification of polymorphic DNA) PCR and REP (repetitive element palindromic) PCR experiments highlighted a similar electrophoretic profile. When genes belonging to the core genome of L. garvieae were tested through a MLRT (multilocus restriction typing), we again observed that all L. garvieae isolates coming from sand bedding and milk shared a common profile, distinguishable from previously studied representative L. garvieae strains. These data indicate that the L. garvieae isolated from sand bedding and milk originated from a few strains adapted to persist in the same habitat. This supports the hypothesis that sand bedding can represent a reservoir of L. garvieae strains and be a potential vehicle for their dissemination in dairy farms.

Identification of Multidrug-Resistant Escherichia coli from Bovine Clinical Mastitis Using a Ceftiofur-Supplemented Medium.

Escherichia coli causes a significant number of clinical mastitis cases in dairy cattle worldwide. The antimicrobial susceptibility of E. coli is important for both human and animal health. Surveillance reports recorded that the efficacy of most antibiotics is substantially preserved but detection of E. coli from clinical mastitis cases producing extended-spectrum beta-lactamases and plasmid-encoded AmpC beta-lactamases has been reported. These resistance determinants have frequently been associated with multidrug resistance. The aim of this study was to determine if a MacConkey agar medium supplemented with 8 mg/L of ceftiofur (MC-CEF) could be a useful tool to identify cephalosporin-resistant and multidrug-resistant (MDR) E. coli among bovine mastitis isolates. During the period 2010-2011, 773 E. coli were isolated from bovine clinical mastitis milk samples collected in 80 dairy farms in Northern Italy. A total of 105 E. coli were selected and assigned either to group randomly selected E. coli (RSEC; n = 53), based on a random selection among the whole collection of 773 E. coli, or to group ceftiofur-resistant E. coli (CEFREC; n = 52). CEFREC isolates were identified by spreading the 773 E. coli isolates on MC-CEF. Minimum inhibitory concentration (MIC) was used to test the phenotypic antimicrobial susceptibility to 16 antibiotics. The MIC results confirmed the ceftiofur resistance in 73.1% (38/52) of CEFREC isolates, whereas all RSEC isolates were susceptible to ceftiofur. The comparison of MIC values for each antibiotic tested between the two groups revealed significantly higher frequencies of resistance to antimicrobials other than ceftiofur in the CEFREC group. Resistance profiles highlighted a significantly higher frequency of MDR isolates among CEFREC (73.1%) than RSEC (17%) E. coli. The results showed that MC-CEF may be a useful selective medium to identify cephalosporin-resistant and MDR E. coli on dairy farms, without performing MIC on all the isolates.

Milk cathelicidin and somatic cell counts in dairy goats along the course of lactation.

This research communication reports the evaluation of cathelicidin in dairy goat milk for its relationship with the somatic cell count (SCC) and microbial culture results. Considering the limited performances of SCC for mastitis monitoring in goats, there is interest in evaluating alternative diagnostic tools. Cathelicidin is an antimicrobial protein involved in innate immunity of the mammary gland. In this work, half-udder milk was sampled bimonthly from a herd of 37 Alpine goats along an entire lactation and tested with the cathelicidin ELISA together with SCC and bacterial culture. Cathelicidin and SCC showed a strong correlation (r = 0.72; n = 360 milk samples). This was highest in mid-lactation (r = 0.83) and lowest in late lactation (r = 0.61), and was higher in primiparous (0.80, n = 130) than in multiparous goats (0.71, n = 230). Both markers increased with stage of lactation, but cathelicidin increased significantly less than SCC. In addition, peak level in late lactation was lower for cathelicidin (5.05-fold increase) than for SCC (7.64-fold increase). Twenty-one (5.8%) samples were positive to bacteriological culture, 20 for coagulase-negative staphylococci and one for Streptococcus spp.; 18 of them were positive to the cathelicidin ELISA (85.71% sensitivity). Sensitivity of SCC >500 000 and of SCC >1 000 000 cells/ml was lower (71.43 and 23.81%, respectively). Therefore, the high correlation of cathelicidin with SCC during the entire lactation, along with its lower increase in late lactation and good sensitivity in detecting intramammary infection (IMI), indicate a potential for monitoring subclinical mastitis in dairy goats. However, based on this preliminary assessment, specificity should be improved (40.41% for cathelicidin vs. 54.57 and 67.85% for SCC >500 000 and >1 000 000 cells/ml, respectively). Therefore, the application of cathelicidin for detecting goat IMI will require further investigation and optimization, especially concerning the definition of diagnostic thresholds.

A Longitudinal Case Study on Dissemination of ST398 Methicillin-Resistant Staphylococcus aureus Within a Dairy Cow Herd.

The study was conducted to describe the dynamics of ST398 methicillin-resistant Staphylococcus aureus (MRSA) on a dairy herd in northeastern Italy. MRSA was first identified in this herd of 120 cows in 2016, after which the herd was sampled once every 3 months for 1 year (April 2016-May 2017). Samples collected included nasal swabs and milk samples from cows and nasal swabs from farmworkers. In addition, pen fencing and teat milk liners were swabbed and air samples from cow pens and the milking parlor were collected. All samples were tested for MRSA using a selective medium; positive isolates were confirmed by mecA PCR. A representative set of MRSA isolates was genotyped using spa typing and multilocus sequence typing. Overall, 34 (mean 23%, range 16-30%) milking cows were found harboring MRSA in the mammary gland and only 6 recovered from infection or colonization. The mean incidence rate was 14% (range 8-20%), mean cure rate was 23% (range 13-43%), and estimated basic reproduction number (R0) was 1.08. The average of positive quarters found was 35.1% and most of the positive quarters (82.4%) developed subclinical mastitis. The mean duration of MRSA colonization in quarters during the study was 247 days, but quarters affected by subclinical mastitis harbored MRSA for a longer time than healthy ones (285 days vs. 131 days). After the second sampling, the farmer segregated MRSA-positive cows from the uninfected cows and milked them last. Despite segregation, 25 newly infected or colonized cows were detected. MRSA isolates from cows, environment, and two farmworkers belonged to the same sequence type (ST398) and spa type (t034). This study highlights the ability of ST398 MRSA to cause a persistent infection of the mammary gland and to survive in the farm environment.

Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus.

To differentiate between the contribution of mammary epithelial cells (MEC) and infiltrating immune cells to gene expression profiles of mammary tissue during early stage mastitis, we investigated in goats the in vivo transcriptional response of MEC to an experimental intra mammary infection (IMI) with Staphylococcus aureus, using a non-invasive RNA sampling method from milk fat globules (MFG). Microarrays were used to record gene expression patterns during the first 24 hours post-infection (hpi). This approach was combined with laser capture microdissection of MEC from frozen slides of mammary tissue to analyze some relevant genes at 30 hpi. During the early stages post-inoculation, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signalling pathway and initiate a sharp induction of innate immune genes predominantly associated with the pro-inflammatory response. At 30 hpi, MEC express genes encoding different acute phase proteins, including SAA3, SERPINA1 and PTX3 and factors, such as S100A12, that contribute directly to fighting the infection. No significant change in the expression of genes encoding caseins was observed until 24 hpi, thus validating our experimental model to study early stages of infection before the occurrence of tissue damage, since the milk synthesis function is still operative. This is to our knowledge the first report showing in vivo, in goats, how MEC orchestrate the innate immune response to an IMI challenge with S. aureus. Moreover, the non-invasive sampling method of mammary representative RNA from MFG provides a valuable tool to easily follow the dynamics of gene expression in MEC to search for sensitive biomarkers in milk for early detection of mastitis and therefore, to successfully improve the treatment and thus animal welfare.

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.

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.

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.

Staphylococcus aureus coa gene sequence analysis can prevent misidentification of coagulase-negative strains and contribute to their control in dairy cow herds.

Accurate and precise differentiation of staphylococci isolated from milk is of importance for udder health management. In particular, the rapid and specific identification of Staphylococcus aureus plays an essential role in the prevention and treatment programs for bovine mastitis. Plasma gelatinization in coagulase assays is routinely used to discriminate S. aureus from other species by detecting the presence of extracellular free staphylocoagulase. However, rarely occurring coagulase-deficient S. aureus strains can be responsible for clinical and subclinical mastitis cases. By investigating S. aureus isolates from a single herd over a 10-year period we identified the persistence of a phenotypically coagulase-negative S. aureus strain and pinpointed the possible cause to a single base pair deletion in the coa gene sequence. Our results support the need to integrate primary biochemical tests with molecular/sequence analysis approaches for correctly identifying and discriminating atypical S. aureus in bovine herds, as the coagulase test alone may fail to detect persistent mastitis-causing strains.

Approach to Selective Dry Cow Therapy in Early Adopter Italian Dairy Farms: Why Compliance Is So Important.

Selective dry-cow therapy (SDCT) became mandatory in Italy on 28 January 2022. During 2020, a group of farms involved in a milk quality program began a pilot experiment with SDCT in order to understand its challenges and to identify areas for procedural improvements. The aim of this study was to describe the challenges and results of the SDCT in early adopters' herds with a special focus on treatment compliance. Retrospective data from 1911 cows from 11 dairy herds were evaluated. Somatic cell counts, clinical mastitis (CM) history, and the California Mastitis Test (CMT) were used as criteria for SDCT. Based on the dairy herd improvement test results and CM history, 48% of all cows should have received antibiotic treatments and internal teat sealants. Adding the CMT at dry-off increased the percentage of antibiotic-treated cows to 62%, with relevant variation among farms. Concerning treatment compliance, 21% of the cows were "non-compliant", suggesting the importance of monitoring treatment compliance. In conclusion, even if commonly used selection criteria for antibiotic treatments were used, the need for more education and in-depth monitoring of the SDCT adoption process was clearly identified. Close collaboration and agreement between veterinarians and farmers are key for SDCT adoption success.

Association between Mastitis Occurrence in Dairy Cows and Bedding Characteristics of Compost-Bedded Pack Barns.

Compost-bedded pack barns (CB) are receiving increasing attention as a housing system that can potentially improve the welfare of dairy cows. This study characterized the frequency and profile of pathogens isolated from clinical (CM) and subclinical (SCM) mastitis in dairy cows housed in CB. It evaluated the association between mastitis occurrence and bedding characteristics in CB systems. Over six months, seven dairy herds were visited monthly for milk and bedding sample collections. Milk samples from mastitis cases were submitted to microbiological identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS). Bedding samples were submitted to physical-chemical (pH, organic matter, moisture, and carbon to nitrogen ratio) and microbiological counting (total bacterial counts, coliforms, streptococci, and staphylococci) analyses. Regression analysis was used to determine the association between mastitis occurrence and CB characteristics. Our results showed that Escherichia coli and environmental streptococci were the most frequently isolated pathogens from CM cases, while Staphylococcus chromogenes and contagious pathogens (Staphylococcus aureus and Streptococcus agalactiae) were the most commonly isolated from SCM cases. Bedding moisture content was positively associated with the incidence of CM. The bedding carbon to nitrogen ratio was negatively associated with the incidence of SCM, and the bedding total bacteria counts tended to be associated with the incidence of SCM. Bedding counts of coliforms positively associated with the prevalence of SCM. Our results can support decision-makers in the dairy industry seeking strategies for bedding management and mastitis control.

Response of the goat mammary gland to infection with Staphylococcus aureus revealed by gene expression profiling in milk somatic and white blood cells.

BACKGROUND: S. aureus is one of the main pathogens responsible for the intra-mammary infection in dairy ruminants. Although much work has been carried out to understand the complex physiological and cellular events that occur in the mammary gland in response to S. aureus, the protective mechanisms are still poorly understood. The objectives of the present study were to investigate gene expression during the early response of the goat mammary gland to an experimental challenge with S. aureus, in order to better understand the local and systemic response and to compare them in two divergent lines of goat selected for high and low milk somatic cell scores. RESULTS: No differences in gene expression were found between high and low SCS (Somatic Cells Score) selection lines. Analysing the two groups together, an expression of 300 genes were found to change from T0 before infection, and T4 at 24 hours and T5 at 30 hours following challenge. In blood derived white blood cells 8 genes showed increased expression between T0 and T5 and 1 gene has reduced expression. The genes showing the greatest increase in expression following challenge (5.65 to 3.16 fold change) play an important role in (i) immune and inflammatory response (NFKB1, TNFAIP6, BASP1, IRF1, PLEK, BATF3); (ii) the regulation of innate resistance to pathogens (PTX3); and (iii) the regulation of cell metabolism (CYTH4, SLC2A6, ARG2). The genes with reduced expression (-1.5 to -2.5 fold) included genes involved in (i) lipid metabolism (ABCG2, FASN), (ii) chemokine, cytokine and intracellular signalling (SPPI), and (iii) cell cytoskeleton and extracellular matrix (KRT19). CONCLUSIONS: Analysis of genes with differential expression following infection showed an inverse relationship between immune response and lipid metabolism in the early response of the mammary gland to the S. aureus challenge. PTX3 showed a large change in expression in both milk and blood, and is therefore a candidate for further studies on immune response associated with mastitis.

Genome characterization and population genetic structure of the zoonotic pathogen, Streptococcus canis.

BACKGROUND: Streptococcus canis is an important opportunistic pathogen of dogs and cats that can also infect a wide range of additional mammals including cows where it can cause mastitis. It is also an emerging human pathogen. RESULTS: Here we provide characterization of the first genome sequence for this species, strain FSL S3-227 (milk isolate from a cow with an intra-mammary infection). A diverse array of putative virulence factors was encoded by the S. canis FSL S3-227 genome. Approximately 75% of these gene sequences were homologous to known Streptococcal virulence factors involved in invasion, evasion, and colonization. Present in the genome are multiple potentially mobile genetic elements (MGEs) [plasmid, phage, integrative conjugative element (ICE)] and comparison to other species provided convincing evidence for lateral gene transfer (LGT) between S. canis and two additional bovine mastitis causing pathogens (Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. dysgalactiae), with this transfer possibly contributing to host adaptation. Population structure among isolates obtained from Europe and USA [bovine = 56, canine = 26, and feline = 1] was explored. Ribotyping of all isolates and multi locus sequence typing (MLST) of a subset of the isolates (n = 45) detected significant differentiation between bovine and canine isolates (Fisher exact test: P = 0.0000 [ribotypes], P = 0.0030 [sequence types]), suggesting possible host adaptation of some genotypes. Concurrently, the ancestral clonal complex (54% of isolates) occurred in many tissue types, all hosts, and all geographic locations suggesting the possibility of a wide and diverse niche. CONCLUSION: This study provides evidence highlighting the importance of LGT in the evolution of the bacteria S. canis, specifically, its possible role in host adaptation and acquisition of virulence factors. Furthermore, recent LGT detected between S. canis and human bacteria (Streptococcus urinalis) is cause for concern, as it highlights the possibility for continued acquisition of human virulence factors for this emerging zoonotic pathogen.

Gut microbiome modifications over time when removing in-feed antibiotics from the prophylaxis of post-weaning diarrhea in piglets.

Post-weaning diarrhea (PWD) in pigs has mainly an infectious basis and control strategies are centred on antibiotics added to the diet. Given concerns on the spread of multi-resistant bacteria, it is necessary to develop alternative prophylactic approaches to control PWD in piglets. The most promising alternative strategies are based on substances that act indirectly on the bacteria by stimulating the immune system or by improving gut health. The aim of this study was to evaluate the effect on the gut microbiota of feed supplemented with a mixture of essential oils (garlic and oregano) in weaning piglets, compared to traditional PWD management (in-feed antibiotics) and to a control group without any diet supplementation. The study involved 197 piglets from 18 litters in a single farm. The piglets were followed from birth to day 58 of age and were weaned at day 26. During the experimental period, the animals were monitored for weight and growth, average daily gain, morbidity and mortality. For the metataxonomics analysis, rectal samples were collected from 17 piglets from the three experimental groups at 4 different time-points (days 1, 12, 26 and 58). Results revealed that the gut microbiota in pre- and post-weaning piglets was dominated by the phyla Firmicutes (51%), Bacteroidetes (25%) and Proteobacteria (16%), which together make up for over 90% of the entire piglet core gut microbiota. The core microbiota comprised 10 taxa before weaning and 43 taxa after weaning, with 7 taxa overlapping between timepoints: two of them (Prevotella 9, p-value = 0.00095; Solobacterium p-value = 0.00821) were significantly more abundant after weaning. All alpha diversity indexes were significantly different between pre- and post-weaning, while only Shannon and Simpson diversity and equitability were significantly different between treatments. Based on the matrix of Bray-Curtis dissimilarities, samples showed clear clustering per timepoint (before and after weaning, p-value < 0.001) and between treatments by timepoint (p-value = 0.0086). The oil-diet group showed a consistently higher F:B ratio at all timepoints. These results show that the pig gut microbiota changes significantly with weaning, and suggest that the use of essential oils as feed supplementation to control PWD does not seem to alter sgnificantly the microbiota nor the growth parameters of piglets, however modifications of specific taxa may occur.

Understanding Antibiotic Resistance as a Perceived Threat towards Dairy Cattle through Beliefs and Practices: A Survey-Based Study of Dairy Farmers.

Antibiotic use is an important component in dairy herd management both to treat bacterial diseases and to maximize animal welfare. However, there is concern among scientists that antibiotic misuse and/or overuse by farmers might promote the emergence of resistant pathogens. We conducted a cross-sectional web-based questionnaire study with dairy farmers/managers in New York, USA to evaluate their (i) level of concern about antibiotic resistance and (ii) interest in adopting new judicious antibiotic use practices regarding mastitis treatment. A total of 118 responses were subjected to statistical analysis. The findings revealed that nearly half (45%) of study participants were undecided or disagreed that antibiotic resistance due to antibiotic use in dairy farming may negatively impact the health of dairy cattle. In contrast, the majority (78%) of participants self-reported that they do not treat with antibiotics at the first sign of mastitis, and the majority (66%) have either fully or partially implemented culture-based mastitis treatment on their farm. The self-reported adoption of culture-based mastitis treatment practices was statistically significantly associated with higher numbers of injectable and intramammary doses of antibiotics used on the participants' farms. These findings will aid future research investigations on how to promote sustainable antibiotic use practices in dairy cattle.

Relationship between total and differential quarter somatic cell counts at dry-off and early lactation.

Mastitis is a most common disease of dairy cows and causes tremendous economic loss to the dairy industry worldwide. Somatic cell counts (SCC) reflect the inflammatory response to infections and is a metric used as key indicator in mastitis screening programs, typically within the framework of national milk recording schemes. Besides the determination of total SCC, the differentiation of cell types has been described to be beneficial for a more definite description of the actual udder health status of dairy cows. Differential somatic cell count (DSCC) represents the combined proportion of polymorphonuclear leukocytes (PMN) and lymphocytes expressed as a percentage of the total. The aim of this study was to investigate the relationship between SCC and differential somatic cell count (DSCC) in individual quarter milk samples collected at different time points: at dry-off, after calving and at the lactation peak. We used individual quarter data from farms representing the specialized production system of Parmigiano Reggiano cheese in Northern Italy. Average DSCC values ranged between 44.9% and 56.3%, with higher values (60.4%-72.1%) in milk samples with ≥ 1 million SCC/ml (where the proportion of samples with DSCC > 70% can be as high as 0.73). Moderate overall correlations between DSCC and log(SCC) were estimated (Pearson = 0.42, Spearman = 0.38), with a clear increasing trend with parity and around the lactation peak (e.g. Pearson = 0.59 at 60 DIM in parity 4). Taking SCC values as indicators of subclinical mastitis, DSCC would diagnose mastitis with 0.75 accuracy. Data editing criteria do have an impact on results, with stricter filtering leading to lower correlations between log(SCC) and DSCC. In conclusion DSCC and SCC provide different descriptions of the udder health status of dairy cows which, at least to some extent, are independent. DSCC alone doesn't provide more accurate information than SCC at quarter level but, used in combination with SCC, can be of potential interest within the framework of milk recording programs, especially in the context of selective dry-cow therapy (SDCT). However, this needs further investigation and updated threshold values need to be selected and validated.