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Abstract Intramammary dry-off treatment is widely considered an effective method for preventing and curing intramammary infection (IMI) in lactating cows; however, it is not commonly used in small ruminants like goats. Therefore, this study was designed to evaluate the effect of an approved cefazolin-based intramammary treatment on the milk microbiota of Alpine dairy goats during the dry and early lactation periods. Sixty goats were randomly selected based on bacteriological results and randomly allocated into the control group (CG) or the treatment group (TG). Cefazolin 250 mg (Cefovet A, Dopharma, Firenze, Italy) was administered to the TG group at dry-off, whereas the CG received no treatment. Pooled milk samples were collected at dry-off (T1; 52 samples), colostrum (T2; 46 samples), and 5–10 days in milk (T3; 55 samples) for bacteriological analysis, somatic cell count (SCC), and 16 S rRNA gene sequencing. SCC levels were initially high in both groups at T1 (TG: 1,588,000 cells/mL; CG: 1,629,000 cells/mL), which significantly decreased at T3 (TG: 148,000 cells/mL; CG: 153,000 cells/mL). Notably, the TG had fewer infected mammary glands than the CG at T3 (p = 0.0248), while no differences were found at T1 or T2. Despite the reduction in SCC and infection rates, cefazolin-based treatment did not significantly affect the alpha- and beta-diversity between the TG and CG. On the other hand, shifts in microbial composition, including fluctuations in Firmicutes, Proteobacteria, and Actinobacteria, were primarily due to the lactation stage rather than the treatment. Differential abundance analyses identified non-pathogenic genera, such as Acinetobacter, Bacteroides, and Paracoccus, that varied between groups at different timepoints. The study provided insights into the effects of cefazolin-based dry goat treatment on goat milk microbiota and its changes during the lactation cycle, demonstrating its potential to reduce SCC and mammary gland infections without significant alterations to the milk microbiota.

Abstract Background Different organic and inorganic bedding materials can be used in dairy farms. Among organic materials, there is an increasing interest in alternative substrates based on recycled manure solids (RMS). Microbiological analyses are crucial to monitor the microbial load and evaluate the presence of pathogens impacting animal welfare and health. However, logistic factors may hamper the possibility of immediately sending fresh samples to the laboratory, requiring storage in cooled conditions before analysis. Methods We assessed the impact of sample refrigeration and freezing of different organic and inorganic bedding substrates including separated raw manure solids (SRMS), anaerobically digested manure solids (ADMS), and new sand (NS), on the total bacterial count (TBC) and on different microbial classes. Results The TBC was higher in fresh NS and ADMS than in refrigerated and frozen samples of the same substrates; in addition, the TBC of ADMS was higher in refrigerated than frozen samples. The TBC of SRMS did not change significantly with refrigeration and freezing. Freezing reduced the total Gram-negative bacterial count more than refrigeration in all substrates. In fresh NS, Gram-negatives were higher than in both refrigerated and frozen NS. Escherichia coli counts were significantly lower in frozen than in refrigerated SRMS. However, both refrigeration and freezing of ADMS resulted in no E. coli growth. The coliform counts were also lower in frozen than refrigerated NS and SRMS. Frozen NS and ADMS showed lower counts compared to refrigeration for Gram-negative bacteria other than E. coli and coliforms. On the other hand, cold storage did not significantly impact the streptococci and streptococcus-like organisms (SSLO) count of all evaluated bedding substrates. Conclusion Refrigeration and freezing affect the bacteriological results of bedding substrates, with freezing generally leading to lower counts than refrigeration. Whenever possible, preference should be given to analyzing fresh bedding samples, however, when necessary, refrigeration would be recommended over freezing, while acknowledging that the measured bacterial load might underestimate the actual microbial content.

ABSTRACT Conventional Mycoplasma spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of Mycoplasma bovis. Despite a wide range of carbon dioxide (CO2) supplementation reported in literature, specific impacts of CO2 on Mycoplasma spp. growth remain unexplored. Our objective was to assess the effect of CO2 concentration on growth detection rates of 24 Mycoplasma spp. isolates from dairy cows. These isolates, mainly M. bovis, were incubated at 37°C in triplicate and three dilution ranges under three CO2 conditions: ambient air or 5% CO2 or 10% CO2. Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log10 cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO2, with less variation observed in ambient air compared with 5% or 10% CO2. However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO2, or 10% CO2. Consequently, Mycoplasma spp. isolated from dairy cattle demonstrated growth after the recommended 7–10 days of culture, even in the absence of supplemental CO2. Given the expected concentration of M. bovis in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being M. bovis, we recommend expanding CO2 concentration ranges in M. bovis culture from 10% CO2 to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO2.IMPORTANCECurrent Mycoplasma spp. culture protocols lack empirical derivation concerning carbon dioxide (CO2) supplementation and are primarily based on the initial cultivation of Mycoplasma bovis. This study indicates that the suitable range for CO2 supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7–10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO2, or 10% CO2 supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing Mycoplasma spp. under ambient air conditions in a laboratory setting.

IntroductionLegionnaires’ disease is a severe pneumonia predominantly caused by Legionella pneumophila (Lp), whose major reservoirs are artificial water systems. As most human infections are caused by L. pneumophila serogroup 1 (Lp1), a reliable method for Lp distinction can be crucial for bacterial spread prevention. As the ability to withstand in environments and to cause the waterborne disease is strongly related to specific genes, the identification of virulent strains can be of great relevance to implement water environmental monitoring and to contain harmful outbreaks to public health. We aimed to test an assay for Lp identification among different Legionella species, and to determine the serogroups. Additionally, we investigated the carriage of virulence and antimicrobial resistance genes.MethodsA total of 90 Legionella spp. isolates identified by phenotypic tests were subjected to the designed quantitative PCR assay targeting specific mip for Lp, wzm for Lp1, pvcA and ahpD for biofilm production. Eleven serogroups were investigated in all our isolates tested positive for mip gene, subsequently analyzed for 12 virulence and 8 antimicrobial resistance genes.ResultsOnly the 70 Lp isolates were positive for mip. Out of 27 Lp isolates belonging to serogroup 1 based on agglutination test, 23 (85.2%) carried wzm. The presence of ahpD and pvcA was found in 94.3 and 98.6% of Lp isolates, respectively. By multiplex PCR, all 23 wzm-positive strains were confirmed as serogroup 1 that was the most predominant (33%). At least one virulence gene was detected in all Lp isolates. The most frequent gene was ispE (100%), followed by issD (96%), icmK and enhC (93%), cpxA (91%), rtxA2 (74%), lvhB8-B9 (61%), and prpA (54%). The other genes were less diffused in Lp strains (rtxA1, 44%; lvhB3-B4, 47%; pvcB, 27%; lvrE, 24%). Of the macrolide resistance genes, the ereA was found in 84% of Lp strains, while only 14 (20%) harbored the lpeAB among the efflux pump genes.ConclusionThe assays validated in this study enable the simultaneous Lp and Lp1 detection. The differentiation of Lp strains according to their virulence properties could be useful to predict the bacterial ability to survive and to cause the disease.

Abstract 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.

Bedding materials are aimed at providing a safe and comfortable resting environment for cows. Control of pathogen proliferation in these substrates is crucial to prevent intramammary infections in dairy cows, as these can significantly impact milk quality, cow health, and farm productivity. This is particularly relevant in the case of organic bedding substrates, including manure-derived materials. This study aimed to evaluate the in vitro effect of a lime-based conditioner (LBC), composed of CaCO3MgCO3 and Ca(OH)2*Mg(OH)2, at increasing concentrations on the physical-chemical characteristics and bacterial counts of untreated anaerobically digested manure solids (ADMS) and separated raw manure solids (SRMS). Unused ADMS and SRMS were evaluated at four LBC weight-based concentrations: 0 (as untreated control), 10, 15, and 20% of LBC inclusion. The bedding materials were assessed immediately after LBC addition (0 h) and after 24, 72, and 168 h of storage at 28°C. The dry matter content (DM), and pH were measured for all the time points. Standard microbiological methods were used to assess total bacterial counts (TBC), other Gram-negative bacteria, coliforms, Escherichia coli, and streptococci and streptococci-like organism (SSLO). It was observed a linear increase in both DM and pH with increasing concentrations of LBC. Specifically, for each percentage unit increase of LBC, the DM of ADMS and SRMS increased by 0.73 and 0.71%, respectively. Similarly, for each percentage unit of LBC, the pH of ADMS and SRMS increased by 0.15 and 0.19, respectively. Conversely, a linear decrease in TBC, Gram-negative bacteria, coliforms, E. coli, and SSLO was observed with increasing concentrations of the LBC. Manure-derived materials without the inclusion of the LBC had bacterial counts that tended to remain high or increase over time. Otherwise, bedding materials with LBC application had reduced bacterial counts. Based on the results of the present study, it was observed that the higher the concentration of LBC, the more significant the reduction of bacterial counts. Specifically, bacterial recovery was lower when higher concentrations of LBC were applied. Our findings underscore the potential of LBC in effectively controlling environmental bacteria and improving the physical-chemical characteristics of manure-derived bedding materials to improve cow health and welfare.

IntroductionA common thought is that extensive and organic breeding systems are associated with lower prevalence of infections in livestock animals, compared to intensive ones. In addition, organic systems limit the use of anti-microbial drugs, which may lead to lower emergence of antimicrobial resistances (AMR).MethodsTo examine these issues, avoiding any a priori bias, we carried out a systematic literature search on dairy cattle breeding. Search was targeted to publications that compared different types of livestock farming (intensive, extensive, conventional, organic) in terms of the circulation of infectious diseases and AMR.Results and discussionA total of 101 papers were finally selected. These papers did not show any trend in the circulation of the infections in the four types of breeding systems. However, AMR was more prevalent on conventional dairy farms compared to organic ones. The prevalence of specific pathogens and types of resistances were frequently associated with specific risk factors that were not strictly related to the type of farming system. In conclusion, we did not find any evidence suggesting that extensive and organic dairy farming bears any advantage over the intensive and conventional ones, in terms of the circulation of infectious agents.

[This corrects the article DOI: 10.1371/journal.pone.0262199.].

ABSTRACT: 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.

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.

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.

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.

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.

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.

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.

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.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provides accurate species-level identification of many, microorganisms retrieved from bovine milk samples. However, not all those microorganisms are pathogenic. Our study aimed to: (1) determine the species-specific prevalence of microorganisms identified in bovine milk of apparently healthy lactating quarters vs. quarters with clinical mastitis (CM); and (2) map current information and knowledge gaps on udder health relevance of microorganisms retrieved from bovine milk samples. A mixed study design (meta-analysis and mapping review) was chosen. We gathered several large Canadian, US and Brazilian data sets of MALDI-TOF results for organisms cultured from quarter milk samples. For meta-analysis, two datasets (apparently healthy quarters vs. CM samples) were organized. A series of meta-analyses was conducted to determine microorganisms’ prevalence. Then, each species reported was searched through PubMed to investigate whether inflammation (increased somatic cell count (SCC) or signs of CM) was associated with microorganism’s recovery from milk. A total of 294 different species of microorganisms recovered from milk samples were identified. Among 50,429 quarter-milk samples from apparently healthy quarters, the 5 most frequent species were Staphylococcus chromogenes (6.7%, 95% CI 4.5–9.2%), Aerococcus viridans (1.6%, 95% CI 0.4–3.5%), Staphylococcus aureus (1.5%, 95% CI 0.5–2.8%), Staphylococcus haemolyticus (0.9%, 95% CI 0.4–1.5%), and Staphylococcus epidermidis (0.7%, 95% CI 0.2–1.6%). Among the 43,924 quarter-milk CM samples, the 5 most frequent species were Escherichia coli (11%, 95% CI 8.1–14.3%), Streptococcus uberis (8.5%, 95% CI 5.3–12.2%), Streptococcus dysgalactiae (7.8%, 95% CI 4.9–11.5%), Staphylococcus aureus (7.8%, 95% CI 4.4–11.9%), and Klebsiella pneumoniae (5.6%, 95% CI 3.4–8.2%). When conducting the PubMed literature search, there were 206 species identified by MALDI-TOF for which we were not able to find any information regarding their association with CM or SCC. Some of them, however, were frequently isolated in our multi-country dataset from the milk of quarters with CM (e.g., Citrobacter koseri, Enterococcus saccharolyticus, Streptococcus gallolyticus). Our study provides guidance to veterinarians for interpretation of milk bacteriology results obtained using MALDI-TOF and identifies knowledge gaps for future research.

The cows receiving antibiotics for intra-mammary infection (IMI) produce milk that cannot be marketed. This is considered waste milk (WM), and a convenient option for farmers is using it as calf food. However, adding to the risk of selecting resistant bacteria, residual antibiotics might interfere with the gut microbiome development and influence gastrointestinal health. We assessed the longitudinal effect of unpasteurized WM containing residual cefalexin on calf intestinal health and fecal microbiota in an 8-week trial. After 3 days of colostrum, six calves received WM and six calves received bulk tank milk (BM) for 2 weeks. For the following 6 weeks, all 12 calves received milk substitute and starter feed. Every week for the first 2 weeks and every 2 weeks for the remaining 6 weeks, we subjected all calves to clinical examination and collected rectal swabs for investigating the fecal microbiota composition. Most WM calves had diarrhea episodes in the first 2 weeks of the trial (5/6 WM and 1/6 BM), and their body weight was significantly lower than that of BM calves. Based on 16S rRNA gene analysis, WM calves had a lower fecal microbiota alpha diversity than that in BM calves, with the lowest p-value at Wk4 (p < 0.02), 2 weeks after exposure to WM. The fecal microbiota beta diversity of the two calf groups was also significantly different at Wk4 (p < 0.05). Numerous significant differences were present in the fecal microbiota taxonomy of WM and BM calves in terms of relative normalized operational taxonomic unit (OTU) levels, affecting five phyla, seven classes, eight orders, 19 families, and 47 genera. At the end of the trial, when 6 weeks had passed since exposure to WM, the phyla Bacteroidetes, Firmicutes, and Saccharibacteria were lower, while Chlamydiae were higher in WM calves. Notably, WM calves showed a decrease in beneficial taxa such as Faecalibacterium, with a concomitant increase in potential pathogens such as Campylobacter, Pseudomonas, and Chlamydophila spp. In conclusion, feeding pre-weaned calves with unpasteurized WM containing antibiotics is related to a higher incidence of neonatal diarrhea and leads to significant changes in the fecal microbiota composition, further discouraging this practice in spite of its short-term economic advantages.

As part of the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), a webinar on the topic “Mastitis Treatment in Lactation” was held, in which eight mastitis experts from different European countries (Spain, The Netherlands, Estonia, Ireland, Poland, Finland, Germany, and Italy) presented their treatment approaches for clinical mastitis in lactation. The aim of this study was to compare the therapeutic approaches to identify commonalities and differences. In all eight participating countries, the decision to start treatment is usually made by the veterinarians, while the farm personnel are responsible for treatment administration. Antibiotic treatment is then typically administered intramammarily. The treatment duration often depends on the label instructions and is frequently extended if Staphylococcus aureus or Streptococcus uberis is involved. Administering supportive therapy, especially non-steroidal anti-inflammatory drugs (NSAIDs) is an established practice in all countries. Penicillin is the first-choice drug for the treatment of mastitis in an increasing number of countries. The use of critically important antimicrobials (CIAs) such as quinolones and third- and fourth-generation cephalosporins is at a low level in Finland and The Netherlands. In Estonia, Germany, Italy, and Spain, the use of CIAs is declining and is only allowed if milk samples are analyzed in advance following the legal framework. Systems for monitoring antibiotic use are being introduced in more and more countries. This exchange of different views will help the European countries to move towards a common high standard of antimicrobial stewardship in veterinary medicine.

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.

Lactating cows are routinely treated at dry-off with antibiotic infusions in each quarter for the cure and prevention of pathogenic intramammary infection, which remains the most common disease in dairy herds. This approach is known as blanket dry-cow therapy, usually effective for the prevention and cure of infections, but has been shown to potentially contribute to the emergence and spreading of antibiotic resistant bacterial strains. Exploring the use of non-antibiotic treatments coupled with selective dry-cow therapy is necessary to reduce the risk of antibiotic resistance and potential interference with milk microbiome balance. The impact of selective dry-cow therapy on the physiological milk microbiome needs to be carefully evaluated. In this small-scale trial, five healthy (no mastits, SCC

Antibiotic resistance is a global problem affecting both human and animal health. Ensuring the strategic and effective use of antibiotics is paramount to combatting the emergence and spread of resistance. This study explored New York State (NYS) dairy farmers' perceptions regarding antibiotic use in dairy farming and antibiotic resistance. Dairy farmers' perceptions were assessed through semi-structured, in-person interviews. Twenty interviews with farm owners and/or managers of 15 conventional and five USDA certified organic dairy farms with 40 to 2,300 lactating cows were conducted. Thematic analysis was used to assess, compare and contrast transcripts for farmers' characterization of their beliefs, values, and concerns. Conventional dairy farmers had a low level of concern about the possible impacts of on-farm antibiotic resistance on human health and believed their antibiotic use was already judicious. Generally, they believed their cattle's health would suffer if antibiotic use were further curtailed. Conventional farmers expressed frustration over the possibility of more stringent governmental, milk cooperative, buyer, or marketer requirements for antibiotic use and associated animal welfare in the future. They attributed expanding regulations in part to misinformed consumer preferences, that farmers felt were influenced by the marketing of organic dairy products. Organic dairy farmers were generally more concerned about issues related to antibiotic resistance than conventional farmers. Both conventional and organic farmers placed emphasis on disease prevention through herd health management rather than treatment. In conclusion, the conventional NYS dairy farmers in this study were skeptical of the need for and benefits of reduced antibiotic use on their dairy farms. Interventions for farmers, delivered by a trusted source such as a veterinarian, that provide training about proper antibiotic use practices and information of possible financial benefits of refining antibiotic use may hold promise.

The primary objective of our study was to assess the ability of a vacuum recorder to detect the presence of bimodal milk flow curves in dairy cows compared with a portable milk flow meter. In a cross-sectional study, 241 individual cow milking observations were analyzed. We simultaneously collected (1) individual cow vacuum events during milking using portable vacuum recorders, and (2) individual cow milk flow curves by attaching a portable milk flow meter to the same milking unit. Presence of bimodality was assessed with the vacuum recorder visually (BIMVA) and with the gold standard method of a milk flow meter through automatic detection (BIMLA). Kappa statistics revealed moderate agreement between BIMVA and BIMLA [κ, 95% confidence intervals (95% CI) = 0.59 (0.46–0.71)]. Diagnostic test statistics for BIMVA for detection of bimodality indicated moderate performance for sensitivity [0.65 (0.52–0.76)] and positive predictive value [0.71 (0.58–0.82)] and high values for specificity [0.92 (0.87–0.95)] and negative predictive value [0.93 (0.84–0.93)]. We conclude that milking vacuum dynamics are a suitable measure to assess bimodal milk flow curves in dairy cows.

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 β-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Among more than twenty species belonging to the class Mollecutes, Mycoplasma bovis is the most common cause of bovine mycoplasmosis in North America and Europe. Bovine mycoplasmosis causes significant economic loss in the cattle industry. The number of M. bovis positive herds recently has increased in North America and Europe. Since antibiotic treatment is ineffective and no efficient vaccine is available, M. bovis induced mycoplasmosis is primarily controlled by herd management measures such as the restriction of moving infected animals out of the herds and culling of infected or shedders of M. bovis. To better understand the population structure and genomic factors that may contribute to its transmission, we sequenced 147 M. bovis strains isolated from four different countries viz. USA (n = 121), Canada (n = 22), Israel (n = 3) and Lithuania (n = 1). All except two of the isolates (KRB1 and KRB8) were isolated from two host types i.e., bovine (n = 75) and bison (n = 70). We performed a large-scale comparative analysis of M. bovis genomes by integrating 103 publicly available genomes and our dataset (250 total genomes). Whole genome single nucleotide polymorphism (SNP) based phylogeny using M.agalactiae as an outgroup revealed that M. bovis population structure is composed of five different clades. USA isolates showed a high degree of genomic divergence in comparison to the Australian isolates. Based on host of origin, all the isolates in clade IV was of bovine origin, whereas majority of the isolates in clades III and V was of bison origin. Our comparative genome analysis also revealed that M. bovis has an open pangenome with a large breadth of unexplored diversity of genes. The function based analysis of autogenous vaccine candidates (n = 10) included in this study revealed that their functional diversity does not span the genomic diversity observed in all five clades identified in this study. Our study also found that M. bovis genome harbors a large number of IS elements and their number increases significantly (p = 7.8 × 10−6) as the genome size increases. Collectively, the genome data and the whole genome-based population analysis in this study may help to develop better understanding of M. bovis induced mycoplasmosis in cattle.

Animal health is affected by many factors such as metabolic stress, the immune system, and epidemiological features that interconnect. The immune system has evolved along with the phylogenetic evolution as a highly refined sensing and response system, poised to react against diverse infectious and non-infectious stressors for better survival and adaptation. It is now known that high genetic merit for milk yield is correlated with a defective control of the inflammatory response, underlying the occurrence of several production diseases. This is evident in the mastitis model where high-yielding dairy cows show high disease prevalence of the mammary gland with reduced effectiveness of the innate immune system and poor control over the inflammatory response to microbial agents. There is growing evidence of epigenetic effects on innate immunity genes underlying the response to common microbial agents. The aforementioned agents, along with other non-infectious stressors, can give rise to abnormal activation of the innate immune system, underlying serious disease conditions, and affecting milk yield. Furthermore, the microbiome also plays a role in shaping immune functions and disease resistance as a whole. Accordingly, proper modulation of the microbiome can be pivotal to successful disease control strategies. These strategies can benefit from a fundamental re-appraisal of native cattle breeds as models of disease resistance based on successful coping of both infectious and non-infectious stressors.

The objective of the present study was to investigate the effect of pre-milking mechanical teat stimulation on milk yield and milking performance of dairy buffaloes in early lactation. For this purpose, twenty-two healthy Italian Mediterranean buffaloes in their first to third lactation and in early lactation (

Dry and early lactation periods represent the most critical phases for udder health in cattle, especially in highly productive breeds, such as the Holstein Friesian (HF). On the other hand, some autochthonous cattle breeds, such as the Rendena (REN), have a lower prevalence of mastitis and other transition-related diseases. In this study, milk microbiota of 6 HF and 3 REN cows, all raised on the same farm under the same conditions, was compared. A special focus was placed on the transition period to define bacterial groups' prevalence with a plausible effect on mammary gland health. Four time points (dry-off, 1 d, 7-10 d and 30 d after calving) were considered. Through 16S rRNA sequencing, we characterized the microbiota composition for 117 out of the 144 milk samples initially collected, keeping only the healthy quarters, in order to focus on physiological microbiome changes and avoid shifts due to suspected diseases. Microbial populations were very different in the two breeds along all the time points, with REN milk showing a significantly lower microbial biodiversity. The taxonomic profiles of both cosmopolitan and local breeds were dominated by Firmicutes, mostly represented by the Streptococcus genus, although in very different proportions (HF 27.5%, REN 68.6%). Large differences in HF and REN cows were, also, evident from the metabolic predictive analysis from microbiome data. Finally, only HF milk displayed significant changes in the microbial composition along the transition period, while REN maintained a more stable microbiota. In conclusion, in addition to the influence on the final characteristics of dairy products obtained from milk of the two breeds, differences in the milk microbiome might, also, have an impact on their mammary gland health.

Late lactation is a critical moment for making mastitis management decisions, but in small ruminants the reliability of diagnostic tests is typically lower at this stage. We evaluated somatic cell counts (SCC) and cathelicidins (CATH) in late lactation sheep and goat milk for their relationship with intramammary infections (IMI), as diagnosed by bacteriological culture (BC). A total of 315 sheep and 223 goat half-udder milk samples collected in the last month of lactation were included in the study. IMI prevalence was 10.79% and 15.25%, respectively, and non-aureus staphylococci were the most common finding. Taking BC as a reference, the diagnostic performance of SCC and CATH was quite different in the two species. In sheep, receiver operating characteristic (ROC) analysis produced a higher area under the curve (AUC) value for CATH than SCC (0.9041 versus 0.8829, respectively). Accordingly, CATH demonstrated a higher specificity than SCC (82.92% versus 73.67%, respectively) at comparable sensitivity (91.18%). Therefore, CATH showed a markedly superior diagnostic performance than SCC in late lactation sheep milk. In goats, AUC was

Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB, cna, clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina), while sel was harboured especially in one Mediterranean country (Tunisia). The etb, seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy) confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.