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.

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.

Dairy farmer's income, working time, and antimicrobial use under different dry cow therapy protocols.

Mastitis is one of the most common diseases of dairy cattle. It has a high impact on farm economy, farmers' working time, and antimicrobial usage (AMU). Selective dry cow therapy (SDCT) is an effective means of reducing AMU without negatively affecting udder health. The objective of our study was to evaluate the impact of SDCT implementation on farmer's income, working time, and AMU, using a bioeconomic model. A stochastic dairy simulation model (DairyHealthSim) based on a weekly model was used to simulate herd dynamics, reproduction, milk production, culling decisions, health outcomes, and the management of health events. A specific module was developed for the simulation of quarter-level intramammary infection (IMI) acquisition and elimination during the lactation and dry-off periods, and 25 different farm settings were defined to represent herds with various udder health situations. We then defined 20 scenarios of SDCT by combining both the use of different thresholds of somatic cell count and milk bacteriology for treatment allocation and the use of internal teat sealant (ITS). All SDCT protocols had a low impact on farmer's income, and we identified some protocols with a positive farm gross margin (up to 15.83 CA$/dried- cow). We also found that adding an ITS to all cows led to greater economic gain. The application of SDCT had a low impact on farmers' working time, except when milk bacteriology was used for decision-making. Antimicrobial treatment to all cows above 200,000 cells/mL at last control, with the use of ITS on all cows, seems a good choice in most dairy farms. These findings could be used to convince farmers to adopt this strategy at dry-off.

Redefining On-Farm Practices: The Perceived Impact of a Responsible Antimicrobial Use Regulation on Dairy Farmers.

The global food animal industry faces a growing concern regarding antimicrobial resistance (AMR), primarily driven by the use of antimicrobials (AMs) for the treatment, control, and prevention of diseases. Addressing this challenge requires promoting responsible antimicrobial use (AMU) practices. In 2019, the province of Québec, Canada, took a significant step by implementing a regulation that limits the use of AMs of very high importance for human medicine (category I AMs as defined by Health Canada) in the food animal industry. However, the implementation of such regulation can significantly influence behavioral shifts among producers, contributing to the wider effort against AMR. Therefore, the objective of this observational study was to describe the perceived changes in knowledge of dairy producers and on-farm practices following the implementation of this regulation, using a cohort design. Data collection involved administering questionnaires to 87 dairy producers from 3 regions of the province of Québec (Estrie, Montérégie, Centre-Du-Québec) before (2017-2018) and after (2020-2021) the implementation of the regulation. The questionnaires explored the descriptive characteristics of farms, the knowledge of producers about the categorization of AMs, their on-farm treatment practices, and the perceived impacts of the regulation. Statistical analysis included t-tests and McNemar tests to compare the paired data obtained using the 2 questionnaires. The results indicated an increase in the knowledge score (the number of AMs correctly categorized by the producers by their importance for human medicine) after the implementation of the regulation, suggesting an improved understanding of the categorization of AMs based on their importance for human medicine. Trends in AMU practices for treating clinical mastitis and reproductive diseases suggested that category I AMs were less likely to be reported as the primary treatment after the regulation, while category II AMs were more often reported as primary treatment. Adoption of the selective dry cow therapy method significantly increased, while the use of teat sealants remained unchanged. Moreover, producers had divergent perceptions regarding the effect of the regulation on the cure rates and disease frequencies. This disparity emphasizes the need for comprehensive data collection to discern the risks associated with such regulatory shifts. The study acknowledges several limitations, including the potential for recall bias, confirmation bias, and desirability bias. Despite these limitations, this study shows that implementing regulations to encourage responsible AMU drives positive transformations in producers' knowledge and on-farm practices. This underscores the pivotal impact of proactive interventions in combating the escalating threat of AMR within the global food animal industry.

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.

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.

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.

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.

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.

Use of ATP luminometry to assess the cleanliness of equipment used to collect and feed colostrum on dairy farms.

The objective of this observational cross-sectional study was to describe the cleanliness of various equipment used for colostrum harvest and calf feeding procedures on dairy farms in Québec, Canada. The study was performed on 42 commercial dairy herds also enrolled in another study aiming to determine the transfer of passive immunity over a 14-mo period. Information on colostrum quality (using Brix value) and cleanliness (total aerobic and total coliform count) were recorded as well as various practices focused on colostrum-feeding equipment and preweaning period using a standard questionnaire. During the study period, colostrum and milk-feeding equipment cleanliness was assessed using direct surface swabbing with Hygiena Ultrasnap swabs. A total of 155 swab samples were obtained from 6 pieces of equipment. Adenosine triphosphate collected from the swabbed surface reacts with the luciferase solution present in the swab by bioluminescence, which is proportional to the quantity of ATP present and quantified as relative light units (RLU). The description of feed equipment cleanliness (defined as the maximal RLU found for a specific herd, dichotomized as <1,000 RLU vs. ≥1,000 RLU) was compared with the herds' descriptive characteristics, focusing on the first 2 components of a multiple correspondence analysis. The median (range) RLU for buckets used for colostrum harvest, bucket or bottle used for feeding, tube feeders, milking colostrum line, and internal surface of the nipples were 41 RLU (3-1,625 RLU), 78 RLU (<1-3,765 RLU), 29 RLU (<1-2,177 RLU), 83 RLU (<1-9,968 RLU), and 1,101 RLU (2-9,546 RLU), respectively. The first 2 components of multiple correspondence analysis explained 24.7% of data variances and were related to the farms' hygiene and health (13.0%) and feeding practices (11.7% of data variance). The maximal dichotomized luminometry value (<1,000 RLU or ≥1,000 RLU) was associated with hygiene and health dimension. This study gave promising results concerning the potential application of ATP luminometry for calf rearing practices assessment.

Under-Recognition of Neonatal Acute Kidney Injury and Lack of Follow-Up.

OBJECTIVE: Acute kidney injury (AKI) incidence is 30% in neonatal intensive care units (NICU). AKI is associated with increased mortality and risk of chronic kidney disease (CKD) in children. To assess follow-up and early CKD, we retrospectively reviewed outcomes of Cincinnati Children's Hospital Medical Center (CCHMC) cohort of neonates from the AWAKEN trial (2014). STUDY DESIGN: Data from 81 CCHMC patients were extracted from the AWAKEN dataset. KDIGO (Kidney Disease: Improving Global Outcomes) criteria for serum creatinine (SCr) and urine output (UOP) <1 mL/kg/h, reported per 24 hours on postnatal days 2 to 7, were used to define AKI. Charts were reviewed until May 2019 for death, nephrology consult, AKI diagnosis on discharge summary, follow-up, and early CKD at >6 months of age (defined as: estimated glomerular filtration rate < 90 mL/min/1.73 m2, hyperfiltration, proteinuria, hypertension, or abnormal ultrasound). Patients were considered to have renal follow-up if they had ≥1 follow-up visit containing: SCr, urinalysis, or blood pressure measurement. RESULTS: Seventy-seven patients had sufficient data to ascertain AKI diagnosis. In total 47 of 77 (61%) were AKI+ by SCr or UOP criteria (20 stage 1, 14 stage 2, 13 stage 3). Four died during their admission and five were removed from CKD analyses due to urologic anomalies. AKI-UOP alone outnumbered AKI-SCr (45 AKI+ vs 5 AKI+ for all stages). 33% of patients had <2 SCr measured while inpatient. Only 3 of 47 AKI+ patients had a nephrology consult (all stage 3 by SCr) and 2 of 47 had AKI included in discharge summary. 67% of AKI+ patients had follow-up. In total 10 of 43 (23%) AKI+ versus 12 of 25 (48%) AKI- patients had ≥1 marker of early CKD assessed after 6 months. Based on SCr, 3 of 7 (43%) AKI+ had hyperfiltration versus 0 of 7 (0%) AKI- (p = 0.19). CONCLUSION: AKI is vastly under-recognized in the NICU, especially if based on SCr alone. This leads to insufficient follow-up to ascertain renal sequelae in this high-risk population. KEY POINTS: · AKI is under-recognized in high-risk neonates.. · There is a lack of adequate follow-up.. · Identification of AKI by SCr alone is insufficient..

Scoping review on clinical definition of bovine respiratory disease complex and related clinical signs in dairy cows.

Bovine respiratory disease complex (BRD) is a worldwide multifactorial infectious disease. Antimicrobials are commonly used for treating BRD because bacteria are often involved. The clinical diagnosis of BRD is a challenge, especially in adult dairy cows, where information on this syndrome is scant. Having a definition based on consistent and reliable clinical signs would improve the accuracy of BRD diagnosis and could help to develop an optimal treatment approach by an early detection. The aim of this scoping review was to review clinical signs that could be recognized by producers in dairy cattle suffering from naturally occurring infectious respiratory disease, as reported in the literature. A review of the literature was performed for articles published between January 1, 1990 and January 1, 2020. The search of literature in English, French, and Italian languages included 2 different databases (Pubmed, https://pubmed.ncbi.nlm.nih.gov/; CAB abstract, https://www.cabi.org/publishing-products/cab-abstracts/). Clinical signs were categorized as follows: (1) "general manifestations of disease," which included behavioral changes or fever; (2) "alterations in respiratory function," which included clinical signs specifically associated with the respiratory tract examination; and (3) "clinical signs of other body systems," which included clinical signs related to other systems such as diarrhea or subcutaneous emphysema. The focus of the review was on clinical signs that could be monitored by animal handlers and producers. A total of 1,067 titles were screened, and 23 studies were finally included. The most common general clinical signs were increased body temperature (reported in 83% of studies, n = 19), change in feed intake (26%, n = 6), altered mentation (22%, n = 5), and decreased milk production (17%, n = 4). The alterations in respiratory function noted were nasal discharge (74%, n = 17), cough (65%, n = 15), altered respiratory dynamic or dyspnea (61%, n = 14), increased respiratory rate (43%, n = 10), and ocular discharge or lacrimation (30%, n = 7). The clinical signs associated with infectious respiratory disease reported in the 23 studies generally lacked a clear description of what constitutes a deviation from normality (0-50% of studies clearly reported what was considered normal versus abnormal depending on the clinical signs). This limitation prevented any comparison between studies that apparently reported the same "clinical sign," but possibly referred to a different assessment and definition of what was considered normal versus abnormal. Therefore, the definition of clinical signs in a repeatable way with validated interobserver agreement to determine the optimal combination for the diagnosis of BRD in dairy cows is needed. This could lead to a more judicious use of antimicrobials for respiratory disease in adult dairy cows.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models.

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of ß-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1ß and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.

Evaluation of quarter-based selective dry cow therapy using Petrifilm on-farm milk culture: A randomized controlled trial.

The objective of this randomized controlled trial was to assess the efficacy of an on-farm culture system using Petrifilm (3M, London, ON, Canada) for targeted treatment decisions at the quarter level at dry-off and its effects on dry period intramammary infections (IMI) and udder health and milk production in the subsequent lactation. A total of 568 cows (2,247 quarters) from 9 dairy herds with bulk tank somatic cell count <250,000 cells/mL in Québec, Canada, were systematically enrolled and randomly allocated to 4 groups: 2 quarter-based selective (QSDCT) groups, using results of quarter-milk culture on Petrifilm, and 2 blanket dry cow therapy (BDCT) groups. The 2 QSDCT groups consisted of (1) antimicrobial to infected quarters and internal teat sealant (ITS) to healthy quarters (QSDCT/ITS); and (2) antimicrobial and ITS to infected quarters and ITS to healthy quarters (QSDCT+ITS/ITS). The 2 BDCT groups were (1) antimicrobial alone to all quarters (BDCT); and (2) antimicrobial and ITS to all quarters (BDCT+ITS). Quarter milk samples were collected at dry-off and after calving for routine bacteriological culture at the laboratory to monitor IMI; data on milk production, somatic cell count, and clinical mastitis recorded up to 120 d in milk were retrieved from health and DHI records. The probability of avoiding antimicrobial treatment in QSDCT groups was estimated at 48.3% (95% confidence interval: 35.7, 60.9). There was no significant difference between the 4 treatment groups regarding acquisition of new IMI (15.9, 13.2, 15.8, and 15.1% probability for BDCT, BDCT+ITS, QSDCT/ITS, and QSDCT+ITS/ITS, respectively) or persistence of existing IMI (3.2, 2.1, 3.4, and 2.7% probability, respectively) over the dry period. In the subsequent lactation, there was no difference between groups regarding incidence of clinical mastitis (2.4, 3.7, 2.9, and 1.7% respectively for BDCT, BDCT+ITS, QSDCT/ITS, and QSDCT+ITS/ITS), mean milk somatic cell score (1.7, 2.0, 2.0, and 2.0 respectively), or mean daily milk production (43.8, 44.2, 43.2, and 42.6 kg/d, respectively) during the first 120 d in milk. In conclusion, QSDCT using the Petrifilm on-farm culture system to detect infected quarters at dry-off is an interesting option to decrease antibiotic use without any negative effects on udder health or milk production in the first 120 d of the subsequent lactation compared with BDCT.

Bayesian assessment of diagnostic accuracy of a commercial borescope and of trimming chute exams for diagnosing digital dermatitis in dairy cows.

Digital dermatitis (DD) is a worldwide infectious disease of cattle that causes lameness, discomfort, and economic losses. The reference standard test to diagnose DD is visual observation in a trimming chute, which cannot be practically performed daily on dairy farms. Moreover, some lesion misclassification may occur using this standard diagnostic method. The possibility of misclassification makes the use of a trimming chute debatable as a perfect reference standard test. The objective of this study was, therefore, to assess the diagnostic accuracy of a commercial borescope and trimming chute exam. The accuracy (sensitivity and specificity) of the tests and DD prevalence were investigated using Bayesian latent class analyses. Our hypothesis was that a commercial borescope can be routinely used to diagnose DD in a milking parlor without previous feet cleaning. A cross-sectional study was performed in a freestall facility. The lesions were scored (M0, M1, M2, M3, M4, M4.1) in the milking parlor with a borescope, followed by an examination in the trimming chute 48 to 72 h after the borescope exam. A total of 870 hind feet were scored during 2 sessions of trimming chute exams and borescope exams in the milking parlor. The data were analyzed in 2 ways. First, data were dichotomized into DD lesions (M1, M2, M3, M4, M4.1) and absence of DD lesions (M0). Second, data were dichotomized into active DD lesions (M1, M2, M4.1) and inactive lesions (M0, M3, M4). A Bayesian latent class model allowing for conditional dependence between tests was used to estimate tests' accuracy, likelihood ratio, and DD prevalence. When the data were dichotomized into DD lesions (M1-M4.1) versus absence of DD (M0) lesions, the sensitivity and specificity of the borescope was 55% [95% credible interval (CrI) 40-71%] and 81% (95% CrI 75-88%). The sensitivity of trimming chute exams was 79% (95% CrI 68-88%), and specificity was 80% (95% CrI 71-89%). When the data were dichotomized into active lesions (M1, M2, M4.1) versus inactive lesions or absence of lesions (M3, M4, M0), the sensitivity and specificity of the borescope were, respectively, 32% (95% CrI 13-58%) and 91% (95% CrI 88-95%). The sensitivity and specificity of trimming chute exams were 91% (95% CrI 81-97%) and 81% (95% CrI 75-89%), respectively. In conclusion, it is possible to use the borescope in the milking parlor without cleaning the feet to monitor prevalence of DD lesions. However, an isolated borescope examination, especially for diagnosing active DD lesions, has low sensitivity for use as a surveillance method. For such use, the sensitivity could be improved by repeating the borescope exam on a regular basis.

Staphylococcus debuckii sp. nov., a coagulase-negative species from bovine milk.

A novel type strain, designated SDB 2975T (=CECT 9737T=DSM 105892T), of the novel species Staphylococcus debuckii sp. nov. isolated from bovine milk is described. The novel species belongs to the genus Staphylococcus and showed resistance to tetracycline and was oxidase- and coagulase-negative, catalase-positive, and Gram-stain-positive. Phylogenetic relationships of Staphylococcus debuckii SDB 2975T to other staphylococcal species were inferred from 16S rRNA gene and whole-genome-based phylogenetic reconstruction. The 16S rRNA gene comparisons showed that the strain is closely related to Staphylococcus condimenti (99.73 %), Staphylococcus piscifermentans (99.66 %), Staphylococcus carnosus (99.59 %) and Staphylococcus simulans (98.03 %). Average nucleotide identity (ANI) values between S.taphylococcus debuckii SDB 2975T and its closely related Staphylococcus species were 83.96, 94.5, 84.03 and 78.09 %, respectively, and digital DNA-DNA hybridization (dDDH) values were 27.70, 58.02, 27.70 and 22.00 %, respectively. The genome of Staphylococcus debuckii SDB 2975T was sequenced with PacBio and Illumina technologies and is 2 691 850 bp long, has a G+C content of 36.6 mol% and contains 2678 genes and 80 RNAs, including six copies of each5S rRNA, 16S rRNA and 23S rRNA genes. Biochemical profiling and a newly developed PCR assay enabled differentiation of Staphylococcus debuckii SDB 2975T and three other SDB strains from its closest staphylococcal species. Differentiation was also achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Genes unique to Staphylococcus debuckii were identified and a PCR-based assay was developed to differentiate Staphylococcus debuckii from other staphylococcal species. In conclusion, the results of phylogenetic analysis along with the ANI values <95 %, and dDDH values <70 % from closely related species along with the phenotypic and biochemical characteristics and specific MALDI-TOF profiles demonstrated that Staphylococcus debuckii SDB 2975T represents a novel species within the genus Staphylococcus, named Staphylococcus debuckii sp. nov. (SDB 2975T=CECT 9737T=DSM 105892T).

Use of height-independent baseline creatinine imputation method with renal angina index.

BACKGROUND: The Renal Angina Index (RAI) is a validated screening tool used at 12 h of pediatric intensive care unit (PICU) admission to predict severe acute kidney injury (AKI) on day 3 of PICU stay. A measured or height-imputed baseline serum creatinine (SCr) is required for AKI diagnosis and RAI calculation, yet these are often lacking. We assessed an age-based, height-independent baseline SCr calculation and compared the RAI values employing this method to their historical counterpart. METHODS: An electronic algorithm was implemented to generate RAI score for patients admitted to our PICU. We reviewed 157 consecutive patient records from May 2017, until we cumulated 100 with a valid RAI calculation. We compared RAI scores using the age-based SCr imputation method of Pottel to the historical RAI. Our primary outcome was a difference in the rate of RAI fulfillment (≥ 8) reclassification between methods. RESULTS: Of the first 100 patients, 27 had measured baseline SCr and 73 used height imputation. Only two patients had RAI reclassified with the Pottel method (one in each direction). Being small for age or older were associated with ≥ 25% overestimation of the baseline SCr in 20 patients with the Pottel method compared with height imputation. 15/157 patients had a falsely positive RAI due to lack of measured baseline SCr and height. CONCLUSION: The age-based method to estimate baseline SCr offers a viable height-independent alternative for RAI calculation. While less precise than a height-based approach, this lack of precision rarely leads to reclassification of patient RAI status.

An observational cohort study on persistency of internal teat sealant residues in milk after calving in dairy cows.

Our objectives were to evaluate the prevalence of quarters with an observable internal teat sealant (ITS) plug at first milking following calving and investigate persistency of ITS residues in milk after calving. An observational cohort study was carried out on 557 quarters of 156 cows treated with ITS in 6 farms in Quebec, Canada. The presence of an ITS plug at first milking and ITS residues in milk at each milking were observed by producers. The effects of various factors on the odds of observing an ITS plug and persistency of ITS residues in milk were studied using generalized logistic mixed and generalized negative binomial mixed models, respectively. Milk samples were taken on the day before dry-off and on 2 occasions after calving for bacterial identification to detect intramammary infection (IMI) using bacteriological culture followed by MALDI-TOF identification. The association between the absence of an ITS plug and the presence of new IMI was assessed using a mixed logistic regression model. Internal teat sealant plugs after calving were more often observed in rear quarters and in quarters receiving ITS alone at drying-off versus antimicrobial and ITS. We observed an average (standard deviation) persistency of 4.0 d (2.3 d). When an ITS plug was still present at first milking (83% of quarters), the elimination of ITS residues in milk after calving was significantly longer (4.5 d, on average) compared with 1.2 d when an ITS plug was absent. In cows with an ITS plug at calving, we observed a higher number of days of excretion in older cows. When a plug could not be observed, rear quarters, older cows, and cows with a long dry period duration excreted ITS residues for a significantly longer period. The lack of a significant association between the absence of a plug and the odds of new IMI at calving suggests that despite the loss of the plug, cows were still protected against new IMI. Although we were able to highlight some statistically significant risk factors explaining persistency of ITS residues following calving, observed differences were often relatively small and, perhaps, not clinically relevant. In conclusion, an ITS plug was present until first milking after calving for 83% quarters, quarters without an ITS plug at first milking appeared to have been protected from new IMI, and ITS residues could be observed in milk up to 12 d in milk.

In vitro antibiotic susceptibility and biofilm production of Staphylococcus aureus isolates recovered from bovine intramammary infections that persisted or not following extended therapies with cephapirin, pirlimycin or ceftiofur.

Staphylococcus aureus intramammary infections (IMIs) have low cure rates using standard antibiotic treatment and increasing the duration of treatment usually improves therapeutic success. Chronic IMIs are thought to be caused by bacteria presenting a specific virulence phenotype that includes the capacity to produce greater amounts of biofilm. In this study, antibiotic susceptibility and biofilm production by S. aureus isolates recovered from IMIs that were cured or not following an extended therapy with cephapirin, pirlimycin or ceftiofur for 5, 8 and 8 days, respectively, were compared. An isolate was confirmed as from a persistent case (not cured) if the same S. aureus strain was isolated before and after treatment as revealed by the same VNTR profile (variable number of tandem repeats detected by multiplex PCR). The antibiotic minimal inhibitory concentrations (MICs) were determined for these isolates as well as the capacity of the isolates to produce biofilm. Isolates from persistent cases after extended therapy with cephapirin or ceftiofur had higher MICs for these drugs compared to isolates from non-persistent cases (p < 0.05) even though the antibiotic susceptibility breakpoints were not exceeded. Isolates of the ceftiofur study significantly increased their biofilm production in presence of a sub-MIC of ceftiofur (p < 0.05), whereas isolates from the pirlimycin group produced significantly less biofilm in presence of a sub-MIC of pirlimycin (p < 0.001). Relative antibiotic susceptibility of the isolates as well as biofilm production may play a role in the failure of extended therapies. On the other hand, some antibiotics may counteract biofilm formation and improve cure rates.