Relationship Between Facility Type and Bulk Tank Milk Bacteriology, Udder Health, Udder Hygiene, and Milk Production on Vermont Organic Dairy Farms.

The primary objective of this cross-sectional observational study was to determine whether bulk tank milk quality, udder health, udder hygiene and milk production outcomes were associated with facility type on organic dairies. A secondary objective was to identify other management-related risk factors associated with bulk tank milk quality, udder health, udder hygiene, and milk production on organic dairy herds in Vermont. We aimed to enroll 40 farms, to compare herds using the 2 most common systems (freestalls, tiestalls) for housing organic dairy cattle in the state with those using a bedded pack during the non-grazing season (typically November-May). Two general styles of bedded packs were observed: cultivated bedded packs and untilled deep bedded packs. Due to the limited number of herds using bedded packs to house lactating dairy cattle in Vermont, we combined untilled and cultivated bedded packs to describe udder hygiene, milk quality, and udder health on these loose-housing systems deeply-bedded with organic material. The study was completed on 21 farms (5 bedded packs, 6 freestalls, 10 tiestalls) before interruption due to the COVID-19 pandemic. Data captured from Dairy Herd Improvement Association records from the test closest to the date of the farm visit included average somatic cell score (SCS), standardized 150-d milk (pounds), % cows with current high SCS (SCS ≥ 4.0), % cows with newly elevated SCS (previous SCS < 4.0 to current ≥ 4.0), and % cows with chronically elevated SCS (SCS ≥ 4.0 last 2 tests). Multivariable linear regression models were used to describe outcomes by facility type, but suffered from limited statistical power due to small group sample sizes. Unconditional comparisons failed to find statistically significant differences between farms grouped by facility type in metrics captured from Dairy Herd Improvement Association test data, bulk tank milk somatic cell count (BTSCC) and aerobic culture data, or udder hygiene scores. A secondary analysis was conducted using univariate linear regression to identify associations between herd management factors and outcomes for all 21 farms combined. Although not all differences found were statistically significant in this secondary analysis combining all farms, numeric differences that may be biologically important are reported showing farms with deeper bedding had a lower BTSCC, lower newly elevated SCS, lower chronically elevated SCS, lower elevated current SCS, lower average SCS, and better udder hygiene metrics. Farms with lower mean udder hygiene scores had numerically lower chronically elevated SCS, lower elevated current SCS, and lower average SCS. We could not reject the null hypothesis that milk quality and udder health outcomes did not differ by facility type, and this does not preclude the existence of biological differences in these outcomes between facility types. The current study provides insight on factors affecting bulk tank milk quality, udder health and hygiene measures on organic dairy farms in Vermont. Bedded packs may be a viable option for confinement housing during the winter non-grazing season for pasture-based herds interested in a loose-housing system in the Northeastern US, but more research such as longitudinal studies with a larger sample size is needed to test this hypothesis.

Negatively controlled trial investigating the effects of dry cow therapy on clinical mastitis and culling in multiparous cows.

Blanket dry cow therapy (DCT) is a major contributor to overall antibiotic usage on dairy farms in the United States. With low prevalence of intramammary infections at dry-off in US herds today, alternative DCT approaches have been the focus of much research. We hypothesized that complete cessation of DCT [i.e., use of internal teat sealants (ITS) only at dry-off] could be a practical alternative to blanket DCT in well-managed herds. The objective of this negatively controlled clinical trial was to determine the effects of DCT on clinical mastitis (CM) and removal from the herd during the dry period and the first 200 d of the subsequent lactation in multiparous dairy cows treated with only ITS at dry-off. As a secondary objective, we conducted exploratory analysis to identify subpopulations in the herd (based on parity, previous CM history, and dry-period length) where DCT would not affect postcalving udder health, to generate hypotheses about potential alternative selective DCT programs. The study was conducted in a commercial dairy herd in South Dakota from June 2020 to January 2021. Dry-off sessions (n = 43) were scheduled such that all cows at a given session were dried off using ITS alone (ITS only, n = 20 sessions, n = 1,108 cows) or an intramammary DCT product containing 500 mg of cloxacillin (Dry-Clox, Boehringer Ingelheim) followed by ITS (ITS+ABX, n = 23 sessions, n = 1,331 cows). Culling and CM events were recorded by farm workers who were blinded to the treatment status of cows. Hazard ratios (HR) for the effects of the treatment group on CM and removal from the herd were estimated using multivariable Cox proportional hazards, adjusting for the clustered treatment allocation strategy. Risk of removal from the herd during the dry period was lower in ITS+ABX than ITS-only cows (1.1 vs. 2.7%; HR = 0.45; 95% CI: 0.25 to 0.81). Risk of removal from the herd during the first 200 d of lactation was similar in ITS+ABX and ITS-only cows (17.3 vs. 18.0%; HR = 0.98; 95% CI: 0.82 to 1.18). Risk of CM during the first 200 d of lactation was lower in ITS+ABX cows (6.9%; HR = 0.56; 95% CI: 0.41 to 0.76) compared with ITS-only cows (13.4%). The beneficial effects of DCT on CM and removal from the herd were consistently observed across strata of parity, previous CM history, and dry-period length, indicating that no subpopulations could be identified to withhold DCT. The findings from this study indicate that the omission of DCT from the dry-off procedure, when udder health is not taken into consideration, in multiparous cows can have a negative effect on cow health and welfare. Findings from previous research suggest that culture- or algorithm-guided selective dry cow therapy are likely to be safer approaches to improving antibiotic stewardship.

Invited review: Selective treatment of clinical mastitis in dairy cattle.

Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.

To seal or not to seal following an antimicrobial infusion at dry-off? A systematic review and multivariate meta-analysis of the incidence and prevalence of intramammary infections post-calving in dairy cows.

Teat sealants (TSs) consist of sterile formulations with no antibacterial activity. Alone or in combination with antimicrobial (AM) or non-AM treatments, TSs have been commonly used in dairy cows at dry-off to prevent intra-mammary infections (IMIs) during the dry period. This study aimed to identify and synthesise the available evidence on the efficacy of combining TSs with AM treatments on the incidence and prevalence of IMIs. A comprehensive search of three electronic databases, two relevant conference proceedings, and reference lists of reviews and eligible articles was conducted to retrieve and identify studies that could answer the following question: in dairy cows, how does the efficacy of an AM-TS combination administered at dry-off compare with an AM alone for preventing new IMI? In addition to the general IMIs, bacterial species-specific data were extracted and combined into nine distinct pathogen groups: coagulase-positive and negative staphylococci; S. dysgalactiae; non-dysgalactiae Streptococci; E. coli; non-E. coli Enterobacteriaceae; Corynebacterium spp.; yeast and other frequent mastitis pathogens. The structural relationship between each study's prevalence and incidence, as the new (incidence) and persistent (uncured) infections make up the prevalence, was utilised to approximate a variance-covariance matrix for the within-study correlation between their study-specific log odds ratios (ORs). A bivariate random-effects meta-analysis was employed, utilising the within- and between-study correlations to synthesise both outcomes simultaneously. The risk of bias was assessed using the Cochrane ROBINS-I tool, and the quality of the body of evidence was rated using the GRADE approach. A total of 17 trials (16 studies), providing either IMIs incidence (n = 4), prevalence (n = 3) or both (n = 10), were identified. Overall, quarters infused with AM-TS combinations showed lower odds of new IMIs post-calving (OR=0.70; 95% CI=0.57-0.86; Wald test P < 0.001) than those which received only AMs. Across the pathogen groups, varying levels of reduction of new IMIs were found, where administration of TSs was most effective against S. dysgalactiae (OR=0.47; 95% CI=0.23-0.98), non-dysgalactiae streptococci (OR=0.60; 95% CI=0.49-0.74), E. coli (OR=0.62; 95% CI=0.50-0.77), Corynebacterium spp. (OR=0.68; 95% CI=0.52-0.90) and coagulase-negative staphylococci (OR=0.85; 95% CI=0.76-0.94). However, additional TS infusion did not significantly reduce new IMIs in the remaining pathogen groups. The current meta-analytic evidence supports the efficacy of using TS add-on infusions in dairy cows at dry-off for reducing the incidence and prevalence of IMIs post-calving; however, pathogen group differences should be considered.

Effect of Holstein genotype on ex-vivo interleukin-1ß response to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and heat-killed Gram-negative and Gram-positive bacteria.

Effects of Holstein genotype on interleukin-1ß response were assessed by ex-vivo stimulation of whole blood with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or sonicated, heat-killed Gram-negative or Gram-positive bacteria. Holstein genotypes were unselected Holsteins (UH, n = 14) not subjected to selection pressures since the mid-1960s and contemporary Holsteins (CH, n = 13). Milk yield of UH and CH cows differ by more than 4500 kg/lactation. Whole blood was mixed with 0.01 µg LPS, 10 µg LTA or 2.5 × 106 CFU of sonicated, heat-killed E. coli, K. pneumoniae, S. marcescens, S. aureus, S. dysgalactiae, or S. uberis per mL of blood and incubated (4 h, 37 °C). Plasma IL-1ß was quantified by ELISA and log10-transformed concentrations analyzed with a multivariate linear mixed effects model. Responses to bacteria were greater than responses to LPS or LTA. Responses to LPS, LTA and the Gram-negative stimulants were greater in UH than in CH cows while responses to the Gram-positive bacteria did not differ between Holstein genotypes. In both genotypes, strong correlations were detected among IL-1ß responses to the Gram-negative stimulants and to LTA. There were strong correlations among IL-1ß responses to the Gram-positive bacteria in CH cows but only between S. aureus and S. dysgalactiae in UH cows. The IL-1ß response to S. uberis was highly correlated with responses to all of the Gram-negative stimulants in CH cows but only with E. coli in the UH cows. The reduced immune response could make contemporary cows more susceptible to infection by Gram-negative bacteria. Results confirm selection practices since the mid-1960s have altered immune response in the Holstein, at least to Gram-negative bacteria, and validate the need for additional studies to further evaluate the impacts of these selection practices on immune function in contemporary Holsteins.

Selective dry-cow therapy can be implemented successfully in cows of all milk production levels.

Antibiotic stewardship on dairy farms can be heightened through the implementation of selective dry-cow therapy (SDCT). However, some producers are concerned that this practice may be related to poor udder health outcomes in cows with high milk production at the time of dry-off. The objective of this study was to evaluate if the effect of culture-guided SDCT (Cult-SDCT) and algorithm-guided SDCT (Alg-SDCT) on dry-period intramammary infection (IMI) dynamics and postcalving udder health and performance [when compared with blanket dry-cow therapy (BDCT)] varied according to milk production level before dry-off. Data were compiled from clinical trials conducted in the United States and Canada that compared Cult-SDCT and Alg-SDCT to a positive control, i.e., BDCT. In those trials, cows were enrolled 1-2 d before dry-off, randomized to their dry-cow therapy strategy and followed until 120 d in milk of the subsequent lactation. The number of cows and quarters in the final data set were 1,485 and 5,097, respectively. Measured outcomes included quarter-level antibiotic use at dry-off, quarter-level IMI prevalence after calving, quarter-level dry-period IMI cure risk, quarter-level dry-period new IMI risk, cow-level clinical mastitis and removal from the herd during 1-120 d in milk, and somatic cell count and milk yield during 1-120 DIM. The primary objective of analysis was to investigate if the effect of Cult-SDCT and Alg-SDCT on these outcomes, when compared with BDCT, varied according to milk production level before dry-off. To do this, each cow was classified as having low, mid or high production, based on her milk yield tertile group at the most recent herd test before enrollment (low: <23.7 kg/d, mid: 23.7 to 30.4 kg/d, and high >30.4 kg/d). Multivariable generalized estimating equations were used to estimate risk differences and differences in means, and Cox regression was used to estimate hazard ratios. For Cult-SDCT, the proportion of quarters treated with dry-cow antibiotics within each milk production level were 40.7% (low), 41.7% (mid) and 47.2% (high). For Alg-SDCT, the proportions were 60.6% (low), 38.7% (mid), and 35.1% (high). Measures of udder health were not markedly different when comparing Cult-SDCT to BDCT and Alg-SDCT to BDCT. This was consistently observed in low, mid and high producing cows. In conclusion, the findings from this study indicate that Cult-SDCT and Alg-SDCT can be successfully implemented in cows of all milk production levels.

Selective treatment of nonsevere clinical mastitis does not adversely affect cure, somatic cell count, milk yield, recurrence, or culling: A systematic review and meta-analysis.

Treatment of clinical mastitis (CM) contributes to antimicrobial use on dairy farms. Selective treatment of CM based on bacterial diagnosis can reduce antimicrobial use, as not all cases of CM will benefit from antimicrobial treatment, e.g., mild and moderate gram-negative infections. However, impacts of selective CM treatment on udder health and culling are not fully understood. A systematic search identified 13 studies that compared selective versus blanket CM treatment protocols. Reported outcomes were synthesized with random-effects models and presented as risk ratios or mean differences. Selective CM treatment protocol was not inferior to blanket CM treatment protocol for the outcome bacteriological cure. Noninferiority margins could not be established for the outcomes clinical cure, new intramammary infection, somatic cell count, milk yield, recurrence, or culling. However, no differences were detected between selective and blanket CM treatment protocols using traditional analyses, apart from a not clinically relevant increase in interval from treatment to clinical cure (0.4 d) in the selective group and higher proportion of clinical cure at 14 d in the selective group. The latter occurred in studies co-administering nonsteroidal anti-inflammatories only in the selective group. Bias could not be ruled out in most studies due to suboptimal randomization, although this would likely only affect subjective outcomes such as clinical cure. Hence, findings were supported by a high or moderate certainty of evidence for all outcome measures except clinical cure. In conclusion, this review supported the assertion that a selective CM treatment protocol can be adopted without adversely influencing bacteriological and clinical cure, somatic cell count, milk yield, and incidence of recurrence or culling.

Diagnosing Intramammary Infection: Meta-Analysis and Mapping Review on Frequency and Udder Health Relevance of Microorganism Species Isolated from Bovine Milk Samples.

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.

Improving farm-level antimicrobial stewardship benchmarks by reporting antimicrobial use within the context of both the magnitude of disease pressure and the outcome of therapy.

This manuscript explores a method of benchmarking antimicrobial use within the context of farm level therapeutic incidence (a proxy for disease incidence), and the outcome of that therapy. This is reported both within the same farm over time (2016-2019), as well as evaluated across participating farms. Reporting antimicrobial use in this format addresses multiple primary questions necessary for evaluating on farm antimicrobial stewardship: How much disease is recorded? How much antimicrobial use is recorded? How often are antimicrobials included in therapy for each disease? What is the outcome of therapy? The three primary metrics reported are: therapeutic events per 100 cow years (TE/100CY), antimicrobial regimens per 100 cow years (REG/100CY), and the percent therapeutic success (% Success). Success was defined as: the cow remained in the herd and had no further TE recorded within 30 days of the end of the TE being evaluated. These measures identify opportunities for change on an individual farm, such as improvement in disease prevention, or a change in choices about when to include an antimicrobial in the treatment protocol. Therapeutic outcomes provide additional context, in some instances demonstrating differences in recording practices and case definitions, while in other cases serving to safeguard animal welfare as efforts are made to decrease antimicrobial use in the future. Although developed for farm level reporting, the metrics may also be more broadly summarized to meet future reporting requirements for marketing chain or national level antimicrobial use reports. The process outlined here serves as a prototype to be considered when developing antimicrobial use reporting systems where farm level antimicrobial stewardship is the primary objective.

Invited review: Selective use of antimicrobials in dairy cattle at drying-off.

Administering intramammary antimicrobials to all mammary quarters of dairy cows at drying-off [i.e., blanket dry cow therapy (BDCT)] has been a mainstay of mastitis prevention and control. However, as udder health has considerably improved over recent decades with reductions in intramammary infection prevalence at drying-off and the introduction of teat sealants, BDCT may no longer be necessary on all dairy farms, thereby supporting antimicrobial stewardship efforts. This narrative review summarizes available literature regarding current dry cow therapy practices and associated impacts of selective dry cow therapy (SDCT) on udder health, milk production, economics, antimicrobial use, and antimicrobial resistance. Various methods to identify infections at drying-off that could benefit from antimicrobial treatment are described for selecting cows or mammary quarters for treatment, including utilizing somatic cell count thresholds, pathogen identification, previous clinical mastitis history, or a combination of criteria. Selection methods may be enacted at the herd, cow, or quarter levels. Producers' and veterinarians' motivations for antimicrobial use are discussed. Based on review findings, SDCT can be adopted without negative consequences for udder health and milk production, and concurrent teat sealant use is recommended, especially in udder quarters receiving no intramammary antimicrobials. Furthermore, herd selection should be considered for SDCT implementation in addition to cow or quarter selection, as BDCT may still be temporarily necessary in some herds for optimal mastitis control. Costs and benefits of SDCT vary among herds, whereas impacts on antimicrobial resistance remain unclear. In summary, SDCT is a viable management option for maintaining udder health and milk production while improving antimicrobial stewardship in the dairy industry.

Effect of Holstein genotype on ex-vivo cytokine response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during the periparturient period.

Effects of Holstein genotype on innate immune response were assessed with ex-vivo lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation of whole blood from unselected (UH, n = 10) and contemporary (CH, n = 11) Holsteins that differ in production by more than 4,500 kg/lactation. Blood was collected at -14, 7, 28, and 49 days in milk (DIM), mixed with a pathogen-associated molecular pattern (PAMP) molecule (0.01 or 1.0 µg LPS or 10 or 100 µg LTA per mL blood) and incubated (4 h, 37 °C). Plasma cytokines were quantified by ELISA, log10-transformed and analyzed by repeated measures with DIM as the repeated effect. Cytokine responses increased with PAMP dose and decreased as DIM increased. There was a genotype by LPS dose interaction for IL-1ß as response to the low dose was greater in UH but did not differ between genotypes for the high dose. The IL-1ß response was greater while the IL-6 response to LTA tended to be greater in UH than in CH cows. The more negative energy balance of CH cows did not impact genotype difference in cytokine responses. Results indicate selection since the mid-1960s has decreased ex-vivo, whole blood cytokine response of CH cows to LPS and to LTA.

The microbiome of common bedding materials before and after use on commercial dairy farms.

BACKGROUND: Bovine mastitis is one of the most economically important diseases affecting dairy cows. The choice of bedding material has been identified as an important risk factor contributing to the development of mastitis. However, few reports examine both the culturable and nonculturable microbial composition of commonly used bedding materials, i.e., the microbiome. Given the prevalence of nonculturable microbes in most environments, this information could be an important step to understanding whether and how the bedding microbiome acts as a risk factor for mastitis. Therefore, our objective was to characterize the microbiome composition and diversity of bedding material microbiomes, before and after use. METHODS: We collected 88 bedding samples from 44 dairy farms in the U.S. Unused (from storage pile) and used (out of stalls) bedding materials were collected from four bedding types: new sand (NSA), recycled manure solids (RMS), organic non-manure (ON) and recycled sand (RSA). Samples were analyzed using 16S rRNA sequencing of the V3-V4 region. RESULTS: The overall composition as well as the counts of several microbial taxa differed between bedding types, with Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes dominating across all types. Used bedding contained a significantly different microbial composition than unused bedding, but the magnitude of this difference varied by bedding type, with RMS bedding exhibiting the smallest difference. In addition, positive correlations were observed between 16S rRNA sequence counts of potential mastitis pathogens (bacterial genera) and corresponding bedding bacterial culture data. CONCLUSION: Our results strengthen the role of bedding as a potential source of mastitis pathogens. The consistent shift in the microbiome of all bedding types that occurred during use by dairy cows deserves further investigation to understand whether this shift promotes pathogen colonization and/or persistence, or whether it can differentially impact udder health outcomes. Future studies of bedding and udder health may be strengthened by including a microbiome component to the study design.

Comparing Blanket vs. Selective Dry Cow Treatment Approaches for Elimination and Prevention of Intramammary Infections During the Dry Period: A Systematic Review and Meta-Analysis.

A systematic review and a series of meta-analyses were conducted to investigate the efficacy of selective dry cow antimicrobial treatment (SDCT) (in which only infected quarters/cows were treated with an antimicrobial) compared with blanket dry cow treatment (BDCT) (all quarters/all cows received an antimicrobial, regardless of their infection status). A full detailed protocol was published before initiating this review. Studies reporting on the (1) proportion of untreated quarters or cows when using SDCT, (2) intramammary infection (IMI) incidence risk over the dry period, (3) IMI elimination risk, (4) post-calving IMI prevalence, (5) early lactation clinical mastitis incidence, or (6) subsequent lactation milk yield and somatic cell counts were considered eligible. Thirteen articles representing 12 controlled trials, whether randomized or not, were available for analyses. SDCT reduced the use of antimicrobials at dry off by 66% (95% CI: 49-80). There was no difference in the elimination of existing IMI at dry off, between SDCT and BDCT. Meta-regression showed that the risk of IMI incidence during the dry period, IMI risk at calving, early lactation clinical mastitis risk, and early lactation milk yield and somatic cell counts did not differ between SDCT and BDCT as long as an internal teat sealant (65% bismuth subnitrate) was administered to untreated healthy quarters/cows at dry off. For trials not using internal teat sealants, SDCT resulted in higher risk than BDCT of acquiring a new IMI during the dry period and of harboring an IMI at calving. Lines of evidence strongly support that SDCT would reduce the use of antimicrobials at dry off, without any detrimental effect on udder health or milk production during the 1st months of the subsequent lactation, if, and only if, internal teat sealants are used for healthy, untreated quarters/cows.

Prevalence, antibiotic resistance, virulence and genetic diversity of Staphylococcus aureus isolated from bulk tank milk samples of U.S. dairy herds.

BACKGROUND: Colonization of dairy cows by Staphylococcus aureus (S. aureus), especially those which are multi-drug resistant and toxin producing, is a concern for animal health and well-being as well as public health. The objective of this study was to investigate the prevalence, antibiotic resistance, gene content and virulence determinants of S. aureus in bulk tank milk samples (BTM) from U.S. dairy herds. RESULTS: BTM samples were collected, once in winter and once in summer, from 189 U.S. dairy herds. Of 365 BTM samples cultured, the sample and herd prevalence of S. aureus in BTM was 46.6% (170 of 365 samples) and 62.4% (118 of 189 herds), respectively. Among a subset of 138 S. aureus isolates that were stored for further analysis, 124 were genome sequenced after being confirmed as S. aureus using phenotypic tests. The most commonly identified antimicrobial resistance-associated gene was norA (99.2%) and mecA gene responsible for methicillin resistance (MRSA) was identified in one isolate (0.8%). The most frequently detected putative virulence genes were aur (100%), hlgB (100%), hlgA, hlgC, hlb (99.2%), lukE (95.9%) and lukD (94.3%). In the 53 staphylococcal enterotoxin positive isolates, sen (37.9%), sem (35.5%), sei (35.5%) and seg (33.1%) were the most frequently detected enterotoxin genes. Among the 14 sequence types (ST) and 18 spa types identified, the most common was ST2187 (20.9%) and t529 (28.2%), respectively. The most predominant clone was CC97 (47.6%) followed by CC unknown (36.3%). The single MRSA isolate belonged to ST72-CC8, spa type t126 and was negative for the tst gene but harbored all the other virulence genes investigated. CONCLUSION: Our findings indicated a high prevalence of S. aureus in BTM of U.S. dairy herds, with isolates showing little evidence of resistance to antibiotics commonly used to treat mastitis. However, isolates often carried genes for the various enterotoxins. This study identified predominant genetic clones. Despite lower prevalence, the presence of MRSA and multi-drug resistant strains in BTM poses a significant risk to animal and public health if their number were to increase in dairy environment. Therefore, it is necessary to continuously monitor the use of antibiotics in dairy cows.

Evaluation of the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) system in the detection of mastitis pathogens from bovine milk samples.

MALDI-TOF is a chemistry analytical tool that has recently been deployed in the identification of microorganisms isolated from nosocomial environments. Its use in diagnostics has been extremely advantageous in terms of cost effectiveness, sample preparation easiness, turn-around time and result analysis accessibility. In the dairy industry, where mastitis causes great financial losses, a rapid diagnostic method such as MALDI-TOF could assist in the control and prevention program of mastitis, in addition to the sanitation and safety level of the dairy farms and processing facility. However, the diagnostic strengths and limitations of this test method require further understanding. In the present study, we prospectively compared MALDI-TOF MS to conventional 16S rDNA sequencing method for the identification of pathogens recovered from milk associated with clinical and subclinical bovine mastitis cases. Initially, 810 bacterial isolates were collected from raw milk samples over a period of three months. However, only the isolates (481) having both 16S rDNA sequencing and MALDI-TOF identification were included in the final phase of the study. Among the 481 milk isolates, a total of 26 genera (12 g-postive and 14 g-negative), including 71 different species, were taxonomically charecterized by 16S rDNA at the species level. Comparatively, MALDI-TOF identified 17 genera (9 g-positive and 8 g-negative) and 33 differernt species. Overall, 445 (93%) were putatively identified to the genus level by MALDI-TOF MS and 355 (74%) were identified to the species level, but no reliable identification was obtained for 16 (3.3%), and 20 (4.2%) discordant results were identified. Future studies may help to overcome the limitations of the MALDI database and additional sample preparation steps might help to reduce the number of discordances in identification. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional identification methods for common mastitis pathogens, routinely isolated from raw milk. Thus it's adoption will strengthen the capacity, quality, and possibly the scope of diagnostic services to support the dairy industry.

Antimicrobial use quantification in adult dairy cows - Part 3 - Use measured by standardized regimens and grams on 29 dairies in the United States.

This study describes antimicrobial use in terms of standardized regimens per cow year (REG/CY) and grams per cow year (GMS/CY) for 29 dairies in the United States during the years 2016 and 2017. To explore potential priorities for antimicrobial stewardship programs, these measures were stratified by both disease syndrome and antimicrobial class. Potential confounders of use measurements are discussed and challenges for measure interpretation are identified. When measured as REG/CY, the results indicate that mastitis is the disease syndrome with the greatest contribution to overall antimicrobial use. However, when GMS/CY is measured, metritis, lameness and unknown disease syndromes are also significant contributors. When use is stratified by antimicrobial class, measures of REG/CY indicate the greatest magnitude of use is the cephalosporin class. However, when measures of GMS/CY are stratified by drug class, use within the penicillin class contributes more than any other single class. These differences highlight the need for a more complete understanding of the relationship between antimicrobial use measures and their relationship to antimicrobial resistance selection pressure.

Antimicrobial use quantification in adult dairy cows - Part 1 - Standardized regimens as a method for describing antimicrobial use.

This study describes a process to acquire and convert farm treatment records into a standardized regimen format. Multiple sources of on-farm data were utilized to convert the original treatment records to standardized regimens, enabling the generation of objective, granular descriptions of antimicrobial use. These standard regimen descriptions allow antimicrobial use data to be qualitatively stratified by active substance, disease syndrome treated, use category, and route of administration. Quantitative distributions are available for the grams of active substance per administration, the grams of active substance per regimen, the number of administrations, number of days of therapy and the time frame between the first and last administration. Granular quantification by this method informs future research, surveillance and policy.

Antimicrobial use quantification in adult dairy cows - Part 2 - Developing a foundation for pharmacoepidemiology by comparing measurement methods.

As stewardship programmes seek to influence therapeutic choices and reduce resistance selection pressure, it is essential to understand the multiple factors which may influence any given antimicrobial use measure. This article compares 9 measures of antimicrobial use pertaining to adult cows in US dairy systems based on treatment records standardized to a single standardized regimen (SReg) format. There are 3 fundamental parameters underlying the nine measures: amount of active substance (AMOUNT), number of SRegs (ANIMALS) and treatment time (TIME). Additionally, three use categories were considered in the analysis: control of mastitis (Dry Cow), treatment of detected mastitis (Clinical Mastitis) and treatment of other diseases (Other Treatment). The assumptions necessary for calculation of each of the nine measures are identified. The influence of measure choice on farm rank was explored at the farm level both within and across fundamental parameters. Across all use categories, when measures of TIME and ANIMALS were compared, correlations between measures were greater than 0.91. However, when measures of AMOUNT were compared to either measures of TIME or ANIMALS, the correlation between measures was more variable across use categories (R = 0.31-0.91). These comparisons demonstrate that the selection of antimicrobial use metric can affect which dairies are considered the highest users of antimicrobials. Measurement selection also influences which use category has the greatest potential for use reduction. This indicates that measure selection can influence which use category is prioritized as a target for reduction of antimicrobial use. Utilization of this information may allow those developing dairy antimicrobial stewardship programmes to better understand the potential effects of measurement selection on driving changes in antimicrobial use.

Development and Implementation of a National Center of Excellence in Dairy Production Medicine Education for Veterinary Students: Description of the Effort and Lessons Learned.

The need for consortial programs to provide advanced education in food animal veterinary production medicine has been recognized and lauded for nearly three decades. This article describes one effort to create a dairy production medicine curriculum funded by a United States Department of Agriculture (USDA) Higher Education Challenge Grant. This National Center of Excellence in Dairy Production Medicine Education for Veterinarians is housed at the Dairy Education Center of the University of Minnesota and the project was a collaboration of the University of Minnesota, the University of Illinois, the University of Georgia, and Kansas State University. The article reviews the need for innovative ways to educate students who will optimally serve the dairy industry, provides a broad overview of the process of developing and delivering the eight-week dairy production medicine curriculum, and describes the challenges faced and lessons learned as a result of offering such a program.

Evaluation of rapid culture, a predictive algorithm, esterase somatic cell count and lactate dehydrogenase to detect intramammary infection in quarters of dairy cows at dry-off.

Our objective was to compare four tests to standard milk culture followed by MALDI-ToF in quarters of cows at dry-off. Cows (n = 432) were randomly selected from seven U.S. dairy herds already participating in a multi-site clinical trial in summer 2018. Aseptic foremilk samples were collected from quarters (n = 1728) two days prior to dry-off, and subjected to index and reference tests. The four index tests included rapid culture, a predictive algorithm, an esterase strip test measuring somatic cell count (SCC) and a cow-side lactate dehydrogenase (LDH) test. Rapid culture was performed by inoculating quarter milk samples onto a commercial rapid culture plate. Plates were evaluated by technicians after 30-40 h of incubation at 37 ± 2 °C. Quarters were classified as infected if any bacterial growth was observed. For the algorithm test method, all quarters were classified as infected if the cow met any of the following criteria: 1) any Dairy Herd Improvement Association (DHIA) test with a SCC > 200,000 cells / ml during the current lactation or 2) two or more clinical mastitis cases during the current lactation. Esterase-SCC and cow-side LDH tests involved adding milk to the test strip and reading for color changes. For esterase-SCC and cow-side LDH tests, low (≥250 cells / ml and ≥100 U / L) and high (≥500 cells / ml and ≥200 U / L) thresholds were used to classify quarters as infected or not. Composite samples (4 × 2 mL quarter-milk samples commingled) were also tested for rapid culture, esterase-SCC and cow-side LDH tests, such that if a composite sample was positive, then all quarters contributing to that sample were classified as infected. The reference test was traditional aerobic culture conducted in an accredited laboratory using MALDI-ToF for identification of isolates. Traditional culture was only conducted on quarter-milk samples, and consequently, IMI was always considered at the quarter-level. Unconditional logistic regression was used to estimate sensitivity (SE), specificity (SP), apparent prevalence, positive predictive values (PPV) and negative predictive values (NPV) for each index test. Cohen's Kappa (κ) was used to measure agreement between tests. Algorithm, esterase-SCC and cow-side LDH tests had poor agreement with the reference test (κ ranging from 0.01 to 0.12), while rapid culture had fair agreement (κ = 0.28). No test had concurrently high SE and SP. Negative predictive values were moderate to high for all tests.