DNA methylation haplotype block signatures responding to Staphylococcus aureus subclinical mastitis and association with production and health traits.

BACKGROUND: DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis. RESULTS: Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield). CONCLUSIONS: In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures.

Evaluation of potassium monopersulfate footbath solution for controlling digital dermatitis in lactating dairy cattle. A randomized clinical trial.

Our objective was to assess potassium monopersulfate as a disinfectant used in footbath to control digital dermatitis (DD) in dairy cows. We hypothesized that a potassium monopersulfate solution would control DD. A 180-day randomized negative controlled trial was conducted in a 265-Holstein free-stall facility. Throughout the trial, foot bathing was performed bi-weekly using a split (left vs. right feet) footbath: one tub filled with 1% potassium monopersulfate (treatment), the other with tap water (control). Digital dermatitis lesions were scored during trimming chute examinations of the unwashed hind heels every 90 days using the modified M-scoring system. Digital dermatitis lesions were re-categorized into four variables: 1) inactive; 2) active; 3) any; 4) inactive or absence of DD lesions. Three longitudinal outcomes were characterized: risks of 1) developing a DD lesion; 2) reactivating an inactive DD lesion; 3) development of an inactive or the absence of the DD lesion. A generalized linear model was used to compare the variables and longitudinal outcomes between treated and control groups. Prevalence of active DD lesions increased from 12.5% to 39.9% between days 0 and 90. This significant increase in prevalence justified the discontinuation of the study on day 90 for ethical reasons. There was no statistical difference between treated and control groups for the first outcome (RR: 1.0; 95% CI: 0.62, 1.7), the second outcome (RR: 1.0; 95% CI: 0.62,-1.7); or the third outcome (RR: 0.88; 95% CI: 0.37, 2.1). A 1% potassium monopersulfate footbath solution appears ineffective to control DD in this study.

Adoption and decision factors regarding selective treatment of clinical mastitis on Canadian dairy farms.

As clinical mastitis (CM) treatments are responsible for a large portion of antimicrobial use on dairy farms, many selective CM treatment protocols have been developed and evaluated against a blanket treatment approach of CM cases. Selective treatment protocols use outcomes of diagnostic tests to exclude CM cases from antimicrobial treatment when they are unlikely to benefit. To tailor interventions to increase uptake of selective treatment strategies, a comprehension of current on-farm treatment practices and factors affecting treatment decisions is vital. Two questionnaires were conducted among 142 farms across 5 provinces participating in the Canadian Dairy Network for Antimicrobial Stewardship and Resistance in this cross-sectional study. Self-reported adoption of selective CM treatments by dairy farmers was 64%, with median of 82% of cows treated in those herds using selective treatment. Using logistic regression models, the odds to implement a selective CM treatment protocol increased with a decreasing average cow somatic cell count. No other associations were identified between use of a selective CM treatment protocol and farm characteristics (herd size, CM incidence, province, milking system, and housing system). Three subsets of farmers making cow-level CM treatment decisions were identified using a cluster analysis approach: those who based decisions almost exclusively on severity of clinical signs, those who used various udder health indicators, and farmers who also incorporated more general cow information such as production, age, and genetics. When somatic cell count was considered, the median threshold used for treating was >300,000 cells/mL at the last Dairy Herd Improvement test. Various thresholds were present among those considering CM case history. Veterinary laboratories were most frequently used for bacteriological testing. Test results were used to start, change, and stop treatments. Regardless of protocol, reasons for antimicrobial treatment withheld included cow being on a cull list, having a chronic intramammary infection, or being at end of lactation (i.e., close to dry off). If clinical signs persisted after treatment, farmers indicated that they would ask veterinarians for advice, stop treatment, or continue with the same or different antibiotics. Results of this study can be used to design interventions targeting judicious mastitis-related antimicrobial use, and aid discussions between veterinarians and dairy producers regarding CM-related antimicrobial use.

IgA nephropathy in children with minimal proteinuria: to biopsy or not to biopsy?

BACKGROUND: Tubulointerstitial lesions and glomerular inflammation severity have been shown to correlate with proteinuria in children with IgA nephropathy (cIgAN). However, there is a lack of data regarding severity of histopathologic findings in cIgAN in patients with minimal to absent proteinuria since kidney biopsy indications are not well defined in these cases. METHODS: Twenty-eight cIgAN patients with kidney biopsy from 4 different centers in Paris (France) and Montreal (Canada) with a urine protein/creatinine ratio (UPCr) ≤ 0.03 g/mmol and a normal estimated glomerular filtration rate (eGFR > 90 ml/min/1.73 m2) on the day of kidney biopsy prior to treatment were included. RESULTS: Median age was 11.82 (9.32-13.45) years, and median follow-up was 4 years (2.87-6.53). At time of biopsy, median eGFR was 116 (102.3-139.7) ml/min/1.73 m2, and median UPCr was 0.02 (0.011-0.03) g/mmol. Microscopic or macroscopic hematuria was present in 35.7% and 64.3% of cases, respectively. Kidney biopsy microscopy analysis showed mesangial (M1), endocapillary (E1), or extracapillary (C1) hypercellularity in 53.5%, 32.1%, and 7.1% of patients, respectively. Chronic histological lesions were also present: glomerulosclerosis (S1) in 42.8% and tubular atrophy/interstitial fibrosis in 7.1%. Podocytopathic features were detected in 21.4%. An ACE inhibitor or immunosuppressive therapy (IS) was prescribed in 42.8% and 21.4% of these patients respectively. One-third (35.7%) received no treatment. At last follow-up, median eGFR was 111.9 (90.47-136.1) ml/min/1.73 m2, and median UPCr was 0.028 (0.01-0.03) g/mmol. CONCLUSION: cIgAN with minimal proteinuria at time of biopsy might be linked with acute and chronic glomerular lesions.

Comparison of 2 PCR assays on environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS900 PCR assay validated for MAP detection in sheep has been shown to have a higher sensitivity than a commercial PCR and fecal culture. We have now compared the performance of this in-house IS900 PCR assay with a commercial ISMap02 PCR assay for the detection of MAP DNA in bovine dairy farm environmental samples. We purposefully selected 30 culture-positive, 62 culture-negative, and 62 non-interpretable environmental samples. We applied the IS900 PCR assay directly to the frozen inoculum of these samples. Inocula were incubated in an automated system, and growth was confirmed by an acid-fast bacilli stain and the IS900 PCR assay. Among culture-positive samples before incubation, the IS900 PCR assay yielded significantly more positive results than the ISMap02 PCR assay; however, among culture-negative samples, the IS900 PCR assay yielded positive results both before and after incubation. The ISMap02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation.

Frequency of isolation and phenotypic antimicrobial resistance of fecal Salmonella enterica recovered from dairy cattle in Canada.

Salmonellosis is one of the leading causes of gastrointestinal infections in humans. In Canada, it is estimated that approximately 87,500 cases of salmonellosis occur every year in humans, resulting in 17 deaths. In the United States, it is estimated that 26,500 hospitalizations and 420 deaths occur every year. In dairy cattle, infections caused by nontyphoidal Salmonella enterica can cause mild to severe disease, including enteritis, pneumonia, and septicemia. Our study objectives were to determine the proportion of fecal samples positive for Salmonella in dairy cattle in Canada and determine the resistance pattern of these isolates. We used data collected through the Canadian Dairy Network for Antimicrobial Stewardship and Resistance (CaDNetASR). Pooled fecal samples from preweaning calves, postweaning heifers, lactating cows, and manure storage were cultured for Salmonella, and the isolates were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibilities were determined using the minimum inhibitory concentration test, and resistance interpretation was made according to the Clinical and Laboratory Standards Institute. A 2-level, multivariable logistic regression model was built to determine the probability of recovering Salmonella from a sample, accounting for province, year, and sample source. The proportion of farms with at least one positive sample were 12% (17/140), 19% (28/144), and 17% (24/144) for the sampling years 2019, 2020, and 2021, respectively. Out of the 113 Salmonella isolates, 23 different serovars were identified. The occurrence of Salmonella appeared to be clustered by farms and provinces. The most common serovars identified were Infantis (14%) and Typhimurium (14%). Overall, 21% (24/113) of the Salmonella isolates were resistant to at least one antimicrobial. Resistance to tetracycline was commonly observed (17%); however, very limited resistance to category I antimicrobials (categorization according to Health Canada that includes third-generation cephalosporins, fluoroquinolones, polymyxins, and carbapenems) was observed, with one isolate resistant to amoxicillin and clavulanic acid. The proportion of Salmonella isolates resistant to 2 and 3 antimicrobial classes was 3.5% and 8.8%, respectively. Our study provided valuable information on the proportion of fecal samples positive for Salmonella, the serovars identified, and the associated resistance patterns across CaDNetASR herds, at regional and national levels.

Real-Time Acute Kidney Injury Risk Stratification-Biomarker Directed Fluid Management Improves Outcomes in Critically Ill Children and Young Adults.

Introduction: Critically ill admitted patients are at high risk of acute kidney injury (AKI). The renal angina index (RAI) and urinary biomarker neutrophil gelatinase-associated lipocalin (uNGAL) can aid in AKI risk assessment. We implemented the Trial in AKI using NGAL and Fluid Overload to optimize CRRT Use (TAKING FOCUS 2; TF2) to personalize fluid management and continuous renal replacement therapy (CRRT) initiation based on AKI risk and patient fluid accumulation. We compared outcomes pre-TF2 and post-TF2 initiation. Methods: Patients admitted from July 2017 were followed-up prospectively with the following: (i) an automated RAI result at 12 hours of admission, (ii) a conditional uNGAL order for RAI ≥8, and (iii) a CRRT initiation goal at 10% to 15% weight-based fluid accumulation. Results: A total of 286 patients comprised 304 intensive care unit (ICU) RAI+ admissions; 178 patients received CRRT over the observation period (2014-2021). Median time from ICU admission to CRRT initiation was 2 days shorter (P < 0.002), and ≥15% pre-CRRT fluid accumulation rate was lower in the TF2 era (P < 0.02). TF2 ICU length of stay (LOS) after CRRT discontinuation and total ICU LOS were 6 and 11 days shorter for CRRT survivors (both P < 0.02). Survival rates to ICU discharge after CRRT discontinuation were higher in the TF2 era (P = 0.001). These associations persisted in each TF2 year; we estimate a conservative $12,500 health care cost savings per CRRT patient treated after TF2 implementation. Conclusion: We suggest that automated clinical decision support (CDS) combining risk stratification and AKI biomarker assessment can produce durable reductions in pediatric CRRT patient morbidity.

Gene co-expression in response to Staphylococcus aureus infection reveals networks of genes with specific functions during bovine subclinical mastitis.

Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection.

Intramammary and systemic use of antimicrobials and their association with resistance in generic Escherichia coli recovered from fecal samples from Canadian dairy herds: A cross-sectional study.

Antimicrobial resistance (AMR) in animals, including dairy cattle, is a significant concern for animal and public health worldwide. In this study, we used data collected through the Canadian Dairy Network for Antimicrobial Stewardship and Resistance (CaDNetASR) to: (1) describe the proportions of AMR in fecal E. coli, and (2) investigate the relationship between antimicrobial use (AMU) (intramammary and systemic routes, while accounting for confounding by other variables) and AMR/multidrug resistance (MDR - resistance to ≥ 3 antimicrobial classes) in fecal E. coli from Canadian dairy farms. We hypothesized that an increase of the AMU was associated with an increase in AMR in E. coli isolates. A total of 140 dairy farms across five provinces in Canada were included in the study. Fecal samples from pre-weaned calves, post-weaned heifers, lactating cows, and farm manure storage were cultured, and E. coli isolates were identified using MALDI-TOF MS. The minimum inhibitory concentrations (MIC) to 14 antimicrobials were evaluated using a microbroth dilution methodology. AMU was quantified in Defined Course Dose (DCD - the dose for a standardized complete treatment course on a standard size animal) and converted to a rate indicator - DCD/100 animal-years. Of 1134 fecal samples collected, the proportion of samples positive for E. coli in 2019 and 2020 was 97.1% (544/560) and 94.4% (542/574), respectively. Overall, 24.5% (266/1086) of the E. coli isolates were resistant to at least one antimicrobial. Resistance towards tetracycline was commonly observed (20.7%), whereas resistance to third-generation cephalosporins, fluoroquinolones, and carbapenems was found in 2.2%, 1.4%, and 0.1% of E. coli isolates, respectively. E. coli isolates resistant to two or ≥ 3 antimicrobial classes (MDR) was 2.7% and 15%, respectively. Two multilevel models were built to explore risk factors associated with AMR with AMU being the main exposure. Systemic AMU was associated with increased E. coli resistance. For an increase in systemic AMU equivalent to its IQR, the odds of resistance to any antimicrobial in the model increased by 18%. Fecal samples from calves had higher odds of being resistant to any antimicrobial when compared to other production ages and farm manure storage. The samples collected in 2020 were less likely to be resistant when compared to samples collected in 2019. Compared to previous studies in dairy cattle in North America, AMR in E. coli was lower.

Genome-Wide DNA Methylation and Transcriptome Integration Associates DNA Methylation Changes with Bovine Subclinical Mastitis Caused by Staphylococcus chromogenes.

Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.

Barriers and facilitators to implementing a new regulation restricting antimicrobial use in dairy production in Québec, Canada: A qualitative study.

With the emergence of antimicrobial resistance (AMR), many countries are implementing restrictive regulations to reduce antimicrobial use (AMU) in animal production. Although these measures are effective at the national level, their implementation may generate challenges for producers and veterinarians. The objective of this study was to explore the barriers and facilitators of implementing a new regulation restricting the use of antimicrobials of very high importance for human health in the dairy production sector in the province of Québec, Canada. Individual interviews were conducted with fifteen veterinarians and twenty-seven dairy producers. Thematic analysis was performed based on the COM-B model of behavior change (capability-opportunity-motivation-behavior). Our results indicated that the lack of availability of alternative treatments, the long delays related to diagnostic tests and the fear of economic consequences were major barriers to the implementation of the regulation. A small number of producers also perceived that the regulation negatively impacted the health and wellbeing of their animals. Additionally, participants acknowledged the importance of early education and training to better understand the purpose of the regulation and increase its acceptability. Lastly, most participants reported that they had not only reduced their use of antimicrobials of very high importance for human health following the regulation, but they had also increased preventive practices on their farm. This study reveals that the implementation of restrictive regulations to reduce AMU in animal production can lead to multiple challenges in practice. Our results highlight the need for better communication and training of producers and veterinarians before and during the implementation of similar regulations in the future and underline the importance of measuring the direct and indirect impacts of those regulations on productivity and on animal health and wellbeing.

Development of a Nomogram to Estimate the 60-Day Probability of Death or Culling Due to Severe Clinical Mastitis in Dairy Cows at First Veterinary Clinical Evaluation.

Severe clinical mastitis is a frequent disease of dairy cattle. An effective mean of predicting survival despite treatment would be helpful for making euthanasia decisions in poor prognosis cases. The objective was to develop a nomogram for prediction of death or culling in the 60 days following a severe mastitis episode in dairy cows at first veterinary visit in farm settings. A total of 224 dairy cows presenting severe clinical mastitis and examined for the first time by a veterinarian were included in a prospective study. Clinical and laboratory (complete blood cell count, L-lactate, cardiac troponin I, milk culture) variables were recorded. Animals were followed for 60 days. A nomogram was built with an adaptive elastic-net Cox proportional hazards model. Performances and relevance were evaluated by area under the receiver operating characteristic curve (AUC), Harrell's concordance index (C-index), calibration curve, decision curve analysis (DCA) and misclassification cost term (MCT). The nomogram included: lactation number, recumbency, depression intensity, capillary refilling time, ruminal motility rate, dehydration level, lactates concentration, hematocrit, band neutrophils count, monocyte count, and milk bacteriology. The AUC and C-index showed a good calibration and ability to discriminate. The DCA suggested that the nomogram was clinically relevant. Euthanizing animals having less than 25% probability of survival is economically optimal. It could be used for early euthanasia decisions in animals that would not survive despite treatment. To facilitate the use of this nomogram by veterinarians, a web-based app was developed.

Antimicrobial use and its association with the isolation of and antimicrobial resistance in Campylobacter spp. recovered from fecal samples from Canadian dairy herds: A cross-sectional study.

Campylobacteriosis is one of the most common zoonotic diseases in North America. As opposed to humans, animal infections caused by Campylobacter spp. are often asymptomatic. In this study, data collected through the Canadian Dairy Network for Antimicrobial Stewardship surveillance system were used to determine the proportion of Campylobacter spp. and antimicrobial resistant isolates recovered from dairy cattle herds. Additionally, the association of antimicrobial use (AMU) with fecal carriage and antimicrobial resistance (AMR) of Campylobacter spp. were investigated. Pooled fecal samples from 5 animals from each production phase (pre-weaned calves, post-weaned heifers, lactating cows), and a manure storage sample were collected from 140 dairy herds across Canada. Samples were cultured using selective media, and Campylobacter isolates were speciated using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibilities were determined using the minimum inhibitory concentration test, and interpretation was made according to the Clinical and Laboratory Standards Institute. Two multilevel logistic regression models were used to investigate the association between the AMU with the isolation and antimicrobial resistance in Campylobacter spp. Of 560 samples, 63.8% were positive for Campylobacter spp., and 96% of the participating farms had at least one sample source (i.e., calves, heifers, lactating cows, or manure storage) positive for Campylobacter spp. Overall, 54.3% of the Campylobacter spp. isolates were resistant to at least one antimicrobial. Resistance to tetracycline was observed in 49.7% of the Campylobacter spp. isolates, followed by ciprofloxacin (19.9%) and nalidixic acid (19.3%). The proportion of multi-drug resistant (≥3 antimicrobial classes) Campylobacter spp. isolates was low (0.3%); however, 15.6% were resistant to two different classes of antimicrobials. Samples collected from lactating cows, heifers, and manure storage were more likely to be positive for Campylobacter spp. compared to calves. Total AMU was associated with a decreased probability of recovering Campylobacter spp. In addition, AMR to either tetracycline or ciprofloxacin had an interaction with antimicrobial use. The probability of resistance to tetracycline increased for each unit increase in the total AMU (Defined Course Dose/100 animal-years), while the probability of resistance to ciprofloxacin decreased. Campylobacter coli isolates were more likely to be resistant to ciprofloxacin and tetracycline when compared to C. jejuni. Our study demonstrated that Campylobacter spp. is widespread among Canadian dairy farms, and a higher proportion of resistance to tetracycline was identified. The total AMU was associated with increased resistance to tetracycline in Campylobacter spp. isolates; however, for ciprofloxacin the AMU was associated with decreased resistance.

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.

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.

Clonal and plasmidic dissemination of critical antimicrobial resistance genes through clinically relevant ExPEC and APEC-like lineages (ST) in the dairy cattle population of Québec, Canada.

Antimicrobial resistance can be effectively limited by improving the judicious use of antimicrobials in food production. However, its effect on the spread of AMR genes in animal populations is not well described. In the province of Québec, Canada, a new legislation implemented in 2019 has led to an unprecedented reduction in the use of critical antimicrobials in dairy production. We aimed to investigate the potential link between ESBL/AmpC E. coli isolated before and after legislation and to determine the presence of plasmids carrying genes responsible for critical AMR. We collected fecal samples from calves, cows, and manure pit from 87 Québec dairy farms approximately 2 years before and 2 years after the legislation came into effect. The whole genomes of 183 presumptive ESBL/AmpC E. coli isolated after cefotaxime enrichment were sequenced. Their phylogenetic characteristics (MLST, serogroup, cgMLST) and the presence of virulence and resistance genes and replicons were examined. A maximum likelihood phylogenetic tree was constructed based on single nucleotide polymorphism (SNPs). We identified 10 clonal lineages (same cgMLST) and 7 clones (SNPs ≤ 52). Isolates belonging to these clones could be found on different farms before and after the legislation, strongly suggesting a clonal spread of AMR genes in the population during this 4-year period. All isolates were multidrug resistant (MDR), with clone 2 being notable for the presence of macrolide, fluoroquinolone, and third-generation cephalosporin resistance genes. We also identified clinically relevant ExPEC (ST10) and APEC-like lineages (ST117, ST58, ST88) associated with the presence of ExPEC and APEC virulence genes, respectively. Our data also suggests the presence of one epidemic plasmid belonging to the IncY incompatibility group and carrying qnrs1 and blaCTX-M-15. We demonstrated that AMR genes spread through farms and can persist over a 4-year period in the dairy cattle population through both plasmids and E. coli clones, despite the restriction of critical antimicrobial use. MDR ExPEC and APEC-like STs are present in the normal microbiota of cattle (more frequently in calves). These data increase our knowledge on gene dissemination dynamics and highlight the fact that biosecurity measures should be enhanced in this industry to limit such dissemination.

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.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis.

BACKGROUND: Mastitis caused by different pathogens including Streptococcus uberis (S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due to S. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurring S. uberis subclinical mastitis and healthy control cows (n = 3/group) were studied. RESULTS: Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower in S. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGET P-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S. uberis infection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG. CONCLUSION: The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Integration of the Renal Angina Index and Urine Neutrophil Gelatinase-Associated Lipocalin Improves Severe Acute Kidney Injury Prediction in Critically Ill Children and Young Adults.

Introduction: Acute kidney injury (AKI) occurs in one-fourth of children and young adults admitted to pediatric intensive care unit (PICU). Severe AKI (sAKI; Kidney Disease: Improving Global Outcomes stage 2 or 3) is associated with morbidity and mortality. An AKI risk stratification system, the Renal Angina Index (RAI) calculated at 12 hours of admission, exhibits excellent performance to rule out sAKI at 72 hours of admission. We found that integration of urine neutrophil gelatinase-associated lipocalin (NGAL) with RAI improves prediction of sAKI. We now report the first-year results after implementation of our prospective automated RAI-NGAL clinical decision support (CDS) program. Methods: Patients 3 months to 25 years of age were eligible. Admission order sets have a conditional order for urine NGAL released when a 12-hour RAI ≥8. The primary outcome was sAKI any time at days 2 to 4 of admission. We assessed performance of the RAI and RAI+/NGAL to predict the primary outcome. Results: A total of 1427 unique patients accounted for 1575 admissions. In 147 admissions, RAI was ≥8. RAI <8 had negative predictive value (NPV) of 0.98 (95% CI 0.97-0.99); RAI ≥ 8 had positive predictive value (PPV) of 0.37 (95% CI 0.30-0.46) to predict days 2 to 4 sAKI (area under the receiver operating characteristic curve [AUC-ROC] 0.88 [95% CI 0.84-0.92]). Of 147 RAI+ patients, 89 had NGAL available. RAI/NGAL combination improved PPV (0.64, 95% CI 0.50-0.79) without decrement in NPV (0.98, 95% CI 0.97-0.98). Conclusion: AKI biomarker assessment directed by risk stratification improves prediction of sAKI in critically ill children and young adults. This CDS process has potential to enrich the population for interventional study, although improvement to adherence to CDS is needed.

Impact of a Regulation Restricting Critical Antimicrobial Usage on Prevalence of Antimicrobial Resistance in Escherichia coli Isolates From Fecal and Manure Pit Samples on Dairy Farms in Québec, Canada.

To tackle antimicrobial resistance (AMR), one of the major health threats of this century, the World Health Organization (WHO) endorsed a global action plan in 2015. This plan calls countries to develop national actions to address AMR. The province of Québec, Canada, adopted a new regulation on the 25th of February 2019, to limit the use in food animals of antimicrobials of very high importance in human medicine. We aimed to establish the impact of this regulation by comparing the AMR situation in dairy cattle in Québec ~2 years before and 2 years after its introduction. We sampled calves, cows, and the manure pit in 87 farms. Generic and putative ESBL/AmpC E. coli were tested for susceptibility to 20 antimicrobials. Logistic regression was used to investigate whether the probability of antimicrobial resistance differed between isolates obtained from the pre and post regulation periods by sample type (calves, cows, manure pit) and in general. To identify AMR genes dissemination mechanisms, we sequenced the whole genome of 15 generic isolates. In the generic collection, at the herd level, the proportion of multidrug resistant (MDR) isolates, decreased significantly from 83 to 71% (p = 0.05). Folate inhibitor and aminoglycoside resistances demonstrated a significant decrease. However, when analyzed by sample type (calves, cows, manure pit), we did not observe a significant AMR decrease in any of these categories. In the ESBL/AmpC collection, we did not detect any significant difference between the two periods. Also, the general resistance gene profile was similar pre and post regulation. We identified both clonal and plasmidic dissemination of resistance genes. In conclusion, as early as 2 years post regulation implementation, we observed a significant decrease in MDR in the dairy industry in Quebec in the generic E. coli collection with folate inhibitor and aminoglycoside resistances showing the most significant decrease. No other significant decreases were yet observed.