NE2248: Mastitis Resistance to Enhance Dairy Food Safety, Milk Quality, and Animal Welfare

(Multistate Research Project)

Status: Active

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[02/07/2023]

Date of Annual Report: 02/07/2023

Report Information

Annual Meeting Dates: 11/02/2022 - 11/04/2022
Period the Report Covers: 10/01/2021 - 09/30/2022

Participants

Bill Owens Louisiana Louisiana State University
Pamela Adkins Missouri University of Missouri
John Barlow Vermont University of Vermont
Sandra Godden Minnesota University of Minnesota
Paolo Moroni New York Cornell University
Dave Wilson Utah Utah State University
Pamela Ruegg Michigan Michigan State University

Brief Summary of Minutes

Minutes Attachment:

NE2248 Minutes FY2022.pdf

Accomplishments

OBJECTIVE 1: Characterize host mechanisms associated with mastitis susceptibility, and resistance to improve economic outcomes and animal welfare.   Michigan State University Member: Pamela Ruegg In 2022, MSU researchers explored 2 potential mechanisms for enhancing immune responses to intramammary infection and determined that an alternative (and more practical) dosing schedule for Imrestor (de Campos, et al., AJVR, 2022) was effective in reducing new IMI and may have hastened involution at dry off. We also performed intramammary challenges with Strep uberis to 40 cows, half of whom were supplemented with a pre-biotic yeast fermentation product and used flow cytometry on the milk to evaluate changes in immune and clinical responses.  University of Minnesota Members: S. Godden, E. Royster, L. Caixeta, N. Noyes, B. Crooker Contributors: S. Rowe, P. Gordon Lippolis, J. D., E. J. Putz, T. A. Reinhardt, E. Casas, W. J. Weber and B. A. Crooker. Effect of Holstein genotype on immune response to an intramammary Escherichia coli challenge.   Primiparous unselected (n = 5) and contemporary (n = 7) Holstein cows received 430 cfu of E. coli strain P4 in 1 quarter. Milk bacterial counts, somatic cell count and BSA concentrations, complete blood cell counts, rectal temperature, and serum and milk whey cytokine (IL-1β and IL-6) concentrations were evaluated to assess effects of genotype on mastitis severity. Longitudinal data were analyzed using general linear models with PROC MIXED with day of study as the repeated effect. Whole blood transcriptomes were generated by RNA sequencing. Transcripts with a false discovery rate of P < 0.05 and a delta log2 expression value greater than 0.7 or less than −0.7 were used for functional enrichment analysis. Bacterial counts were consistently greater in milk from contemporary than unselected Holstens from d 0.25 through d 2.5. Milk somatic cell count increased within 6 h (d 0.25) after E. coli administration in both genotypes but did not differ between genotypes after d 1. The number of differentially expressed transcripts in blood at each of the postinfusion sampling times was consistently greater (4- to 90-fold) in contemporary cows. A key difference between the immune reaction of the 2 genotypes was that the immune response to E. coli was largely contained within the mammary gland of the unselected Holsteins but became more systemic in contemporary cows. Results demonstrate unselected Holsteins exerted more effective control of E. coli infused into the mammary gland and thus support the hypothesis that selection practices since the mid-1960s have resulted in contemporary Holsteins with an immune system that is less effective in fighting intramammary infections. Manuscript published in 2022. Brink, A.A., W. J. Weber, J. D. Lippolis, J. B. Cole, S. M. Godden, A. J. Seykora and B. A. Crooker. 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 (n = 10) and contemporary (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, 37ºC). 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-1β as response to the low dose was greater in unselected Holsteins but did not differ between genotypes for the high dose. The IL-1β response was greater while the IL-6 response to LTA tended to be greater in unselected than in contemporary Holsteisn. The more negative energy balance of contemporary Holsteins did not impact genotype difference in cytokine responses. Results indicate selection since the mid-1960s has decreased ex-vivo, whole blood cytokine response of contemporary Holstein cows to LPS and to LTA. Manuscript published in 2022.   Brink, A.A., W. J. Weber, J. D. Lippolis, J. B. Cole, A. K. Rendahl, L. S. Caixeta, S. M. Godden, A. J. Seykora and B. A. Crooker. Effect of Holstein genotype on ex-vivo whole blood interleukin-1β response to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and heat-killed Gram-negative and Gram-positive bacteria.   Effects of Holstein genotype on interleukin-1β response were assessed by ex-vivo, whole blood stimulation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) and heat-killed Gram-negative and Gram-positive bacteria. Blood from unselected Holsteins (n=14) that had not been subjected to selection pressures since the mid-1960s and contemporary Holsteins (n=13) was mixed with 0.01 µg LPS, 10 µg LTA or 2.5 x 106 CFU of 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). Milk yield of the contemporary Holsteins is more than 4,500 kg/lactation greater than that of the unselected Holsteins. Plasma IL-1β was quantified by ELISA, log10-transformed, and analyzed with a multivariate linear mixed effects model. Responses to LPS and LTA were greater in UH than in CH cows. Responses to heat-killed bacteria were greater than responses to either LPS or LTA. There was a genotype by Gram-type interaction as responses to Gram-negative bacteria were greater in unselected Holsteins but did not differ between genotypes for Gram-positive bacteria. In both genotypes, IL-1β response to LPS was highly correlated with response to Gram-negative bacteria and to LTA. Results indicate selection has decreased ex-vivo, whole blood IL-1β response of contemporary Holstein cows to LPS, LTA and Gram-negative bacteria. Manuscript will be submitted in 2022.   <ol> <li>Dean, L.S. Caixeta, N. Noyes, S. Godden, B. Crooker, F. Pena Mosca. Impact of the cow udder microbiome on the biodiversity of milk.</li> </ol> Exploratory project to investigate the microbiome in different parts of the mammary gland, and to better understand how microbiome-host interactions influence response to mastitis challenge. Project is still active. Will be completed by end of 2022.  University of Missouri Members: John Middleton, Pamela Adkins Contributors: Samantha Haw, Paige Isensee The University of Missouri has continued our collaboration with the University of Maryland to evaluate big-data genomics to improve dairy cattle health, including investigating mastitis resistance.  Cornell University Member: Paolo Moroni Contributors: Gloria Gioia We will continue the research program investigating the opportunity to understand the epidemiology of Mycoplasma spp and prevalence in US farms. In collaborations with different group, we investigated genotyping and antimicrobial susceptibility profiling of Streptococcus uberis isolated from a clinical bovine mastitis outbreak in a dairy farm. Molecular characterization contributed to understanding their modes of dissemination, and revealed that Strep. uberis infections could be related to a restricted number of predominant cow-adapted strains.  Ohio State University Member: Benjamin D. Enger Contributors: Kellie M. Enger, Pari H. Baker A study was undertaken that investigated how mastitis in rapidly growing heifer mammary glands affects cellular proliferation and apoptosis of mammary epithelial cells and stromal cells. Mastitis reduced apoptosis in the stromal compartment and increased cellular apoptosis of mammary epithelial cells, both changes expected to reduce future milk production capacity of the udder.   University of Tennessee Member: Oudessa Kerro Dego Contributors: Barbara Gillespie, Benti Gelalcha, Aga Gelgie Gelgie A. E. and O. Kerro Dego. A study to determine differentially expressed host-Pathogen factors associated with host susceptibility or resistance during the early stages of M. bovis -mammary epithelial cells coinfection. Mycoplasma bovis is a highly contagious pathogen of cattle which has multiple predilection sites including udder, lung, uterus, joint, heart and an eye. M. bovis is also notoriously resistant to antimicrobials and effective vaccine is a likely attainable prevention and control strategy against M. bovis diseases. In the face of the widespread economic impact on dairy and beef industries, there are multiple vaccine development efforts to date although important virulence factors yet to be identified. An effort to develop a vaccine might necessitate the prior establishment of novel virulence-associated mycoplasma genes and host immunity-associated genes. we proposed a relatively modern approach that involves a co-incubation of bovine mammary epithelial cells along with M. bovis and characterization of up-regulated or down-regulated M. bovis genes and mammary epithelial cells genes at the mRNA level to determine host-pathogen factors responsible for resistance or susceptibility to M. bovis mastitis. which is anticipated to bring forward certain clues about the important virulence genes playing key roles during infection and pathogenesis.  University of Vermont Members: John Barlow, Feng-Qi Zhao In collaboration with Dr. Jianxin Liu of Zhejiang University, Dr. Zhao’s group in Vermont was involved in a study of the associations of formation of blood neutrophil extracellular traps (NETs) with the increase of the mastitis risk and assessment of mammary health in dairy cows. We found that the formation of NETs in the blood of transition dairy cows was significantly higher in cows with high SCCs than in cows with low SCCs, which may be used as a potential indicator for the prognosis of postpartum mastitis risk and management strategies of perinatal dairy cows. The formation of NETs in blood in transition dairy cows may damage the integrity of the blood-milk barrier and thereby increase the risk for mastitis.   OBJECTIVE 2:  Characterize agents associated with intramammary infections and assess their impact on milk quality and animal welfare   Louisiana State University Member: William E. Owens Identify and evaluate bacteria causing bovine mastitis in Louisiana. Test bacteria for antimicrobial resistance to mastitis therapeutics. Stock mastitis isolates and share with other members for further testing and evaluation. Advise dairy producers and fieldmen on mastitis prevention and control methods.   Michigan State University Member: Pamela Ruegg MSU researchers completed a negatively controlled randomized clinical trial that enrolled 240 cases of non-severe clinical mastitis that had growth on Gram-positive segments of selective media and determined that organisms that the most prevalent organisms were Lactococcus and other non-typical catalase-negative cocci. In-vitro susceptibility of these organisms to approved IMM antibiotics varied from results of Streptotocci and Staphylcocci.  University of Minnesota Members: S. Godden, E. Royster, L. Caixeta, N. Noyes, B. Crooker Contributors: S. Rowe, P. Gordon <ol start="2020"> <li>Pena Mosca, C.C. Florentino, L.S. Caixeta. Applying precision dairy farming and diagnostic technologies to detect mastitis at the time of and following dry off in dairy cows in a field study. The main objective of this study was evaluate accuracy of precision dairy monitoring and diagnostic technologies (i.e. differential somatic cell counter) in collecting data that effectively identifies appropriate animals for dry cow therapy compared to SCC and bacteriology. In this study, milk samples were collected 1d prior to dry-off and 3 and 5 days after calving. Milk samples were used for the measurement of differential SCC, SCC, and milk culture to identify intramammary infection. Cow enrollment and health and performance was completed between fall 2019 and fall 2020. Data analysis is ongoing and manuscripts are being prepared for submission in early 2023.</li> <li>Leonard, L.S. Caixeta, E. Shepley, M. Ruch. Randomized clinical trial evaluating the effect of quorum sensing technology on somatic cell count linear score in early lactation. In this study, cows with somatic cell count linear score (LS) greater than 4 at the first milk tests were allocated to two study groups: treatment group received oral boluses containing quorum quenching products whereas the control group did not receive any treatment. Cows will be followed for 4 consecutive milk tests and LS will be compared between groups. Cow enrollment happened between April and October 2022, follow up period will be completed in February 2023. Data analysis and data report will be completed in 2023.</li> </ol>  University of Missouri Members: John Middleton, Pamela Adkins Contributors: Samantha Haw, Paige Isensee The University of Missouri has collaborated with the University of Montreal and other NE-1748/2248 stations to define intramammary infection using MALDI-TOF identification of bacteria isolated from bovine milk samples. Our group has also studied the prevalence of mastitis pathogens in Jersey cattle and their association with subclinical mastitis. We have also done work to evaluate antibacterial activity of Staphylococcus chromogenes isolates originating from dairy cattle.   University of Tennessee Member: Oudessa Kerro Dego Contributors: Barbara Gillespie, Benti Gelalcha, Aga Gelgie Gelgie, A. E., Gillespie, B. E., Desai, S., Almeida, R., Lysnyansky, I., Shpigel, N. Y., Agga, G. E. and Kerro Dego, O. Identification of Critical Virulence Factors of Mycoplasma bovis by Transposon Mutagenesis and Experimental Infection of Dairy Cows. Mycoplasma bovis mastitis is a highly contagious disease of dairy cows that incurs severe economic losses to dairy farms. To date, there are no effective control and prevention measures for M. bovis mastitis. The major constraint to developing effective intervention tools is limited knowledge of the mechanisms of pathogenesis of M. bovis mastitis. The objective of this study was to identify critical virulence factors of M. bovis to cause mastitis in dairy cows. We created random transposon mutant library of Mycoplasma bovis strain PG45 and tested mutant clones in vitro and in vivo to identify mutant clones that lost the ability to cause mastitis. We found that two mutant clones with reduced pathogenicity under in vitro and in vivo mice mastitis model. We tested the pathogenicity of these two mutants by experimental infection of dairy cows and found that one of the two mutants failed to cause mastitis in dairy cows. This gene product could serve as a potential target for the intervention of M. bovis mastitis in dairy cows.   Gelalcha B. D. B. E. Gillespie and O. Kerro Dego. Prevalence and Molecular Epidemiology of Extended-Spectrum β-lactamases Producing E. coli and K. spp. in East Tennessee Dairy Farms. The extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, such as E. coli and Klebsiella species are identified by the World Health Organization and Centers for Disease Control and Prevention of the United States of America as severe threats to global health due to their rapid spread and multi-drug resistance phenotypes. Recent studies increasingly reported the rise of extended-spectrum beta-lactamases-producing-Enterobacteriaceae in cattle. The U.S. Centers for Disease Control and Prevention also reported a 9% average annual increase in hospitalized patients from community-acquired ESBLs pathogens in six consecutive years (2012 – 2017). However, the sources of ESBL Enterobacteriaceae particularly ESBL-E. coli and -Klebsiella species and mechanisms responsible for their increased emergence are not known. We conducted a cross-study to determine prevalence of ESBL-E. coli and -Klebsiella species in dairy cattle farms and their ESBL genes spread mechanisms among them. From a total of 572 rectal fecal and other samples collected from farms, a total of 233 (40.7%) ESBL-E. coli and 57 (10%) ESBL-Klebsiella species were detected. The prevalence of fecal ESBL-E. coli and  ESBL-Klebsiella species were 47.5% (95% CI: 46.2-49.2) and 7.2 % (95% CI:6.5-8.0), respectively. Both ESBL-E. coli and -Klebsiella species were detected from rectal fecal samples and from all three types of samples (manure, feed, and water) not directly collected from animals. Similarly, ESBL-E. coli and -Klebsiella species were detected in 24.2% (8/33) and 6.1 % (2/33) of bulk tank milk samples, respectively. A total of 230 (99.6%) of 231 E. coli tested for antimicrobial susceptibility were resistant to at least one of the 14 antimicrobial agents. Most of the E. coli isolates (94.4%; 218/231) were multidrug-resistant (MDR), of which 42.6% (n=93) showed co-resistance to at least six classes of antibiotics. About 19% (11/57) of Klebsiella species from feces and other samples were MDR; of which 36.4% (4/11) were resistant to at least six classes of antibiotics, including critically important ones.  University of Vermont Members: John Barlow, Feng-Qi Zhao The Barlow lab extended our work whole genome sequencing staphylococcus isolates obtained from cases of clinical and subclinical mastitis on dairy farms. We developed a hybrid sequencing pipeline that integrates long-read Oxford nanopore and short read Illumina shotgun sequencing to generate closed genomes for more than 100 isolates of S. aureus. We have explored the use of these genomes in a comparative genomics approach to identify sequence variation in virulence factor and antimicrobial resistance genes.   OBJECTIVE 3: Assess and apply new technologies and preventative strategies that advance mastitis control, milk quality and/or reduce antimicrobial usage   Michigan State University Member: Pamela Ruegg We evaluated use of reticulorumen temperature sensing bolus to predict onset of clinical signs after intramammary challenge with Strep uberis. We also created an antimicrobial benchmarking tool that uses electronic records to assess and compare antibiotic usage on dairy farms.  University of Minnesota Members: S. Godden, E. Royster, L. Caixeta, N. Noyes, B. Crooker Contributors: S. Rowe, P. Gordon <ol start="2022"> <li>Rowe, L.S. Caixeta, E. Royster, S. Godden. Clinical trial evaluating the health effects of reducing antibiotic use at dry-off in dairy cows. Randomized clinical trial comparing blanket dry cow therapy and no dry cow therapy in a low input commercial dairy farm. Enrollment of cows started in the summer 2021 and will continue in the spring of 2022. Lactation health and performance for the first 100 DIM will continue until winter 2022. Data analysis and reporting will be completed in 2023.</li> </ol> L.S. Caixeta, S. Godden, W. Knauer, J. Hadrich. G. Cramer. Randomized clinical trial evaluating the effect of administering Bovikalc® dry at dry off on udder health, early lactation health and performance, and economics in dairy cows. Randomized clinical trial investigating the effects of pre dry-off administration of acidogenic bolus on udder health during the dry period and in early lactation. Enrollment of cows was completed in the summer and fall of 2021. Cows were followed until the spring of 2022. Data analysis and reporting will be completed in 2022. Godden, S., S. Wells, E. Royster, B. Crooker. Investigation of the Relationship between Method for Processing Recycled Manure Solids and Pathogen Control on Midwest Dairy Farms. Our objectives were to Investigate if method of recycled manure solids (RMS) processing reduces or eliminates viable mastitis pathogens as well as zoonotic pathogens in RMS. In summer of 2021 samples were collected from 27 dairy premises in MN and WI, which were recruited to achieve a sample of different processing methods including Green (GRN; n=6), Composted (COM; n=3), Digested (DIG; n=9), Hot air dried (DRY; n=2), or a combination of systems such as DIG-DRY (n=6) or DIG-Infrared (IR) (n=1). Premises were visited once in summer 2021 to collect slurry and bedding samples before and after each processing step within the system. Solids samples were submitted to the Laboratory for Udder Health (UMN) for aerobic culture to determine counts of mastitis pathogens. Duplicate slurry and solids samples were also submitted to the WI Vet Diagnostic Lab to test for the presence of Mycobacterium avium subsp. paratuberculosis (MAP; liquid culture with PCR confirmation) and Salmonella spp. (culture). Results showed that ready to use (RTU) solids samples from herds using either anaerobic digestion or secondary processing of RMS had lower mastitis bacteria counts for one or more mastitis pathogen groups, as compared to samples from herds using GRN RMS bedding. However, of all the systems evaluated, the use of a combination of DIG plus a SEC system generally resulted in the lowest bacteria counts in RTU solids. The ability of these processing systems to reduce mastitis pathogen counts may contribute to why these herds observed lower BT SCC as compared to herds using GRN RMS bedding. Results also showed a high proportion of raw slurry and GRN RTU RMS samples were positive for MAP and SAL. Despite small sample sizes for some systems, results show that either DIG or SEC processing (e.g. DRY or COM) can result in a substantial numerical, if not statistical, reduction in risk for a pos. MAP or SAL test. However, DIG alone cannot be counted upon to eliminate either MAP or SAL. No MAP or SAL was detected in RTU solids when a combination of DIG plus SEC processing was used. However, the latter results should be interpreted with caution given the very small sample size. Larger studies are needed to more extensively evaluate the biological and economic impacts for all of these RMS processing systems, and the newer IR systems in particular.   Godden, S. and E. Royster. Pilot Study: Refining a Culture-Guided Selective Dry Cow Therapy Program (SDCT) to Reduce Antibiotic Use on Dairy Farms. We hypothesize that by identifying and selectively treating only IMI caused by SSLO, we can dramatically reduce antibiotic use at dry-off by as much as 90%, while maintaining udder health, cow health and performance in the subsequent lactation (versus blanket DCT). In summer, 2021, we conducted a pilot study in 2 commercial herds to evaluate the effect of implementing a SDCT program that identifies and treats only IMI caused by SSLO on measures of quarter health, cow health and performance and antibiotic use (vs. BDCT). Lab culture of samples was completed in summer 2021. Follow-up of next lactation performance (to 120 DIM) was completed in fall 2021. Data analysis was completed in spring/summer 2022. Results of this pilot study showed that a SSLO-targeting SDCT program resulted in an 85% reduction in AMU at DO, with similar infection dynamics during the dry period as compared to BDCT. Furthermore, there was no difference in udder health or performance (e.g. test day milk production or SCC; clinical mastitis occurrence, herd removal) between cows in the SDCT program as compared to BDCT. If future larger controlled trials can demonstrate that these results are repeatable, then SSLO-targeting SDCT programs could represent an opportunity for dairy producers to further enhance antimicrobial stewardship and reduce costs at dry off, while maintaining cow health.  University of Missouri Members: John Middleton, Pamela Adkins Contributors: Samantha Haw, Paige Isensee The University of Missouri has started to evaluate IMIs in heifers to determine when in the heifer lifecycle these infections first occur. This study is underway and is being conducted using cisternal sampling to determine bacteria that is truly in the mammary gland vs just in the teat canal.  Cornell University Member: Paolo Moroni Contributors: Gloria Gioia Validation of commercial PCR for identification of Mycoplasma spp and Staphylococcus aureus   Oregon State University Member: Massimo Bionaz Contributors: Hunter Ford We tested the role of the combination of selenium-yeast with chicory-plantain silage on the response to subclinical intramammary infection (IMI) in sheep. Among others, we run a large blood profiling, complete blood count, and phagocytosis. Data indicated a clear subclinical response with a mild systemic inflammation, but not large effects of the selenium-yeast or the chicory-plantain silage in the response to IMI.  University of Tennessee Member: Oudessa Kerro Dego Contributors: Barbara Gillespie, Benti Gelalcha, Aga Gelgie Vidlund, J., B. E. Gillespie, G. E. Agga, L. Schneider, S. M. Swanson, K. D. Frady, and O. Kerro Dego. Evaluation of Efficacy of Novel Staphylococcal Surface Protein Vaccines Against Staphylococcal Mastitis in Dairy Cows. We developed and evaluated the efficacies of Staphylococcus aureus surface-associated proteins (SASP) and Staphylococcus chromogenes surface-associated proteins (SCSP) vaccines against naturally occurring Staphylococcus aureus mastitis in dairy cows over 300 days of lactation. We divided a total of 45 Holstein dairy cows from the University of Tennessee East Tennessee AgResearch and Education Center-Little River Animal and Environmental Unit (ETREC-LRAEU) dairy farm into three groups and vaccinated with SASP (n=15), SCSP (n=16) vaccines, or injected with saline and used as unvaccinated control (n=14). Cows were monitored for adverse reactions, change in serum and milk antibody titers, milk yield, milk somatic cell count, mastitis incidence, and Staphylococcus aureus and non-aureus staphylococci isolation in milk over 300 days of lactation. Milk and serum antibody titers were increased in the vaccinated cows compared to control cows. Post-calving, staphylococcal subclinical mastitis incidence was significantly reduced both at the cow (SCSP group) and quarter (SCSP and SASP) levels in the vaccinated cows compared to unvaccinated cows.  <ol> <li>Zeng, J. Vidlund, B. Gillespie, L. Cao, G. E. Agga, J. Lin and O. Kerro Dego.</li> </ol> Evaluation of Immunogenicity of Enterobactin Conjugate Vaccine for the Control of E. coli Mastitis in Dairy Cows Current mastitis control programs, when fully applied, reduce contagious mastitis pathogens, but are less effective against environmental mastitis pathogens such as Escherichia coli. Consequently, environmental mastitis pathogens became a major problem for dairy farms with reduced incidence of contagious mastitis pathogens. The current commercially available E. coli J5 mastitis vaccine reduces production losses and severity of clinical mastitis but does not prevent production losses and clinical disease. Effective vaccine that prevent production losses and clinical disease is needed. Recently, a nutritional immunity approach, which restricts bacterial iron uptake by targeting enterobactin molecule secreted by the bacteria is shown to be a promising approach for the control E. coli mastitis in dairy cows. The immunogenicity and safety of this enterobactin-targeting vaccine in dairy cows are not evaluated. The objective of this study was to determine the immunogenicity and safety of the Ententerobactin conjugate vaccine in dairy cows. We divided a total of 12 Holstein dairy cows from the University of Tennessee East Tennessee AgResearch and Education Center-Little River Animal and Environmental Unit (ETREC-LRAEU) dairy farm into two groups of 6 cows each. We vaccinated one group with Enterobactin conjugate vaccine and injected the other group with phosphate-buffered saline (PBS, pH 7.4) and used as unvaccinated control group. We measured enterobactin-specific serum and milk antibody titers. Results from this study showed that the enterobactin conjugate vaccine induced significantly higher enterobactin-specific serum and milk antibody titers. It did not induce any adverse reaction and had no detrimental effects on milk production in vaccinated cows. No difference in the fecal microbiota structure and diversity between vaccinated and unvaccinated control groups. Therefore, our study demonstrated that Ent conjugate vaccine is immunogenic in dairy cows, and it is a promising vaccine to control E. coli mastitis in dairy cows.  University of Vermont Members: John Barlow, Feng-Qi Zhao A collaborative study between Dr. Jianxin Liu of Zhejiang University and Dr. Zhao’s group in Vermont showed that supplementation with N-carbamoylglutamate during the transition period increases the number of lymphocytes and total antioxidant capacity and reduced the plasma level of malondialdehyde and blood reactive oxygen species. Supplementation with N-carbamoylglutamate improves the functions of neutrophils and reduces inflammation and oxidative stress in dairy cows. The Barlow lab extended our work collaborating with the Neher lab at U of Vermont characterizing the bacterial and fungal community of dairy farm bedding systems using an amplicon sequencing approach supplemented with the ecological assessment of cultured Trichocomaceae isolates (focusing on Penicillium and Aspergillus species) and yeasts (Saccharomycetales). This work has relevance to mastitis control by understanding how the choice of bedding materials and its management represent a potential opportunity to curate the microbial community of the housing environment and influence mastitis risk and milk quality. The Barlow lab also completed a case study of dairy cow hock colonization with Staphylococcus aureus. We identified that the same strains colonizing hocks in a herd suffering from a high frequency of hock lesions were also the strains associated with the sporadic cases of S. aureus mastitis in this herd.   Virginia Tech. Members: Christina Petersson-Wolfe As farm size continues to increase and labor remains a concern for dairy farms, many operations have turned to the use of wearable technologies and in-line milk monitoring tools to aid in the detection of disease. However, little research has been done to properly evaluate how, when and where to use these technologies. The purpose of these research efforts is to examine the ability of these tools to identify cows with mastitis prior to the onset of clinical signs. If we can identify a highly sensitive and specific model for the identification of potentially diseased animals, we can then examine early intervention strategies. The ability to intervene early may lead to more timely therapy, with increased cure rates, less severe mastitis and improved animal well-being. As an industry, we face continued scrutiny about the conditions in which cows are housed and the well-being they are afforded. By examining ways to identify diseased animals sooner and provide therapy to them more quickly, we can provide support to consumers that the well-being of our dairy cows is of utmost importance. Specific project goals include: 1) assess the use of pain mitigation at calving to determine impact on animal well-being and 2) assess the data from wearable technologies to identify disease prior to onset of clinical signs.   WORK PLANNED FOR THE COMING YEAR, LISTED BY OBJECTIVE: OBJECTIVE 1   University of Minnesota Members: S. Godden, E. Royster, L. Caixeta, N. Noyes, B. Crooker Contributors: S. Rowe, P. Gordon <ol> <li>A. Crooker, S. M. Godden, L. Caixeta, A. Seykora, M. Schutz, J. D. Lippolis, J. B. Cole and B. Rosen. Reducing mastitis in the dairy cow by increasing the prevalence of beneficial polymorphisms in genes associated with mastitis resistance.</li> </ol> Our premise is that previous selection practices have successfully increased the presence of genetic polymorphisms associated with increased milk yield but have decreased the presences of polymorphisms associated with disease resistance. We are using contemporary Holsteins and unique unselected Holsteins that have not been subjected to selection since 1964 to determine differences in immune and inflammatory responses and mammary gene expression. We will link differential phenotypic responses with whole genome sequencing to identify polymorphisms that impact mastitis resistance. These polymorphisms could enhance gene-assisted selection efforts to increase mastitis resistance and reduce the use of antibiotics. Analyses of samples from intramammary challenge studies with Escherichia coli strain P4 and Streptococcus uberis strain 0140J continues and additional S. uberis and Staphylococcus aureus strain Newbold challenges will be conducted. Impact of Holstein genotype on the immune response is also being evaluated using whole blood stimulation assays and the measurement of cytokine responses to pathogen-associated molecular pattern (PAMP) molecules and to gram-negative and gram-positive bacteria that commonly cause mastitis is also being assessed. University of Missouri Members: John Middleton, Pamela Adkins Contributors: Samantha Haw, Paige Isensee The University of Missouri will continue to work in collaboration with the University of Maryland to evaluate big-data genomics to improve dairy cattle health, including investigating mastitis resistance.  This work will be done using US dairy genomic databases.   Ohio State University Member: Benjamin D. Enger Contributors: Kellie M. Enger, Pari H. Baker A study is underway that will investigate how mammary growth and development is affected by mastitis during first gestation and a second study will investigate how milk secretory processes are disrupted during mastitis in lactating cows.  University of Tennessee Member: Oudessa Kerro Dego Contributors: Barbara Gillespie, Benti Gelalcha, Aga Gelgie We will continue our transcriptome-based study to determine differentially expressed host-Pathogen factors associated with host susceptibility or resistance during the early stages of in vivo IMI and in vitro bacterial (Staph. aureus, M. bovis, Strep. uberis, E. coli)-mammary epithelial cells coinfection.   University of Vermont Members: John Barlow, Feng-Qi Zhao In next year, Zhao Lab will investigate the effects of LPS and cytokines on energy metabolism and lipid synthesis in bovine mammary epithelial cells and look for ways to reduce these effects.   OBJECTIVE 2   Michigan State University Member: Pamela Ruegg We are collecting clinical case data from commercial farms to use to develop prediction algorithms that can be used to help guide clinical mastitis treatment decisions.  We will also be characterizing virulence factors of Lactococci spp., that are associated with intramammary infection.   University of Minnesota Members: S. Godden, E. Royster, L. Caixeta, N. Noyes, B. Crooker Contributors: S. Rowe, P. Gordon <ol start="2023"> <li>Pena Mosca, C.C. Florentino, L.S. Caixeta. Applying precision dairy farming and diagnostic technologies to detect mastitis at the time of and following dry off in dairy cows in a field study. The main objective of this study was to evaluate accuracy of precision dairy monitoring and diagnostic technologies (i.e. differential somatic cell counter) in collecting data that effectively identifies appropriate animals for dry cow therapy compared to SCC and bacteriology. Data analysis is ongoing and manuscripts are being prepared for submission in early 2023.</li> <li>Leonard, L.S. Caixeta, E. Shepley, M. Ruch. Randomized clinical trial evaluating the effect of quorum sensing technology on somatic cell count linear score in early lactation. Data analysis and data report will be completed in 2023.</li> </ol>  University of Missouri Members: John Middleton, Pamela Adkins Contributors: Samantha Haw, Paige Isensee The University of Missouri will work in collaboration with the University of Vermont to determine predictable molecular patterns of Staphylococcus chromogenes isolates deemed to be chronic high somatic cell count associated, chronic low somatic cell count associated, or teat skin associated. The comparison will be done using bacterial whole genome sequencing, MLST strain typing, and MALDI-TOF fingerprinting. We will also continue working to determine antimicrobial activity of S. chromogenes isolates though in vitro assessment of this organism’s ability to inhibit the grown of S. aureus isolates.  Cornell University Member: Paolo Moroni Contributors: Gloria Gioia We will be investigating the antibacterial activity of Lactococcus garvieae isolates originating from milk bedding and water and antibiotic resistance genes in conventional and organic farms.  University of Tennessee Member: Oudessa Kerro Dego Contributors: Barbara Gillespie, Benti Gelalcha, Aga Gelgie We will be characterizing identified potential virulence factors of Mycoplasma bovis using different methods including specific gene deletion and complementation-based approach. We will also conduct molecular characterization of ESBL E. coli and ESBL Klebsiella spp. and their ESBL genes and associated AMR genes and virulence genes.   Utah State University Member: David Wilson Contributors: Gregory Goodell, Tessa Kelly Continue the evaluation of a preservative for milk, colostrum and milk replacer fed to calves.  Preliminary results have identified two concentrations of the preservative that are associated with significantly reduced bacteria counts over time in comparison to those in untreated post-pasteurized milk to be fed to calves. Other applications in dairy and other food industries have found these concentrations safe including with no withdrawal time for human consumption. The goal is to determine a safe but effective concentration of preservative that can be added to colostrum and milk fed to dairy calves.   University of Vermont Members: John Barlow, Feng-Qi Zhao The Barlow lab will continue our collaboration with Dr. Pamela Adkins at the University of Missouri to explore the epidemiology and pathogenesis of Staphylococcus chromogenes intramammary infections. The Barlow lab will complete whole genome sequencing of Staphylococcus species isolates to identify potential virulence factors and antimicrobial resistance genes and explore the phylogeny and ecology of Staphylococcus species and mobile genetic elements among these species. The Barlow lab will continue to explore the epidemiology of Staphylococcus aureus and non-aureus staphylococci in small to medium sized dairy farms.   OBJECTIVE 3   Michigan State University Member: Pamela Ruegg We are performing a validation trial for antimicrobial benchmarking and will  be adding up to 50 herds to the peer comparison dataset.  We are completing a trial assessing risk factors for presence of methicillin resistant staphylococci in bulk tank milk.  University of Minnesota Members: S. Godden, E. Royster, L. Caixeta, N. Noyes, B. Crooker Contributors: S. Rowe, P. Gordon <ol start="2023"> <li>Rowe, L.S. Caixeta, E. Royster, S. Godden. Clinical trial evaluating the health effects of reducing antibiotic use at dry-off in dairy cows. Randomized clinical trial comparing blanket dry cow therapy and no dry cow therapy in a low input commercial dairy farm. Data analysis and reporting will be completed in 2023.</li> </ol> L.S. Caixeta, S. Godden, W. Knauer, J. Hadrich. G. Cramer. Randomized clinical trial evaluating the effect of administering Bovikalc® dry at dry off on udder health, early lactation health and performance, and economics in dairy cows. Randomized clinical trial investigating the effects of pre-dry-off administration of acidogenic bolus on udder health during the dry period and in early lactation. Data analysis and reporting will be completed in 2022.   University of Missouri Members: John Middleton, Pamela Adkins Contributors: Samantha Haw, Paige Isensee The University of Missouri will continue working to determine when heifer IMIs occur to better focus implementation of prevention strategies. This will be done by collecting FNA/cisternal sampling, gland secretions, and teat canal swabs from heifers to determine if results differ between these collection methods.   Oregon State University Member: Massimo Bionaz Contributors: Hunter Ford Analysis of the transcriptome of the somatic cells from the above study.  University of Tennessee Member: Oudessa Kerro Dego Contributors: Barbara Gillespie, Benti Gelalcha, Aga Gelgie We will conduct controlled experimental vaccination and challenge studies to determine the efficacy of polyvalent vaccine containing staphylococcal surface proteins and streptococcal surface proteins against Staphylococcus aureus and Streptococcus uberis mastitis in dairy cows. Similarly, we will conduct controlled experimental vaccination and challenge studies to determine the efficacy of our novel Enterobactin conjugate vaccine in small groups of dairy cows.   University of Vermont Members: John Barlow, Feng-Qi Zhao The Barlow lab will continue to explore the relationship between housing and bedding management practices and mastitis risk using culture-based and culture independent (metagenomic) methods. Barlow lab will continue to explore the potential role of endogenous inhibitor bacteria and bacteriocins in the epidemiology of Staphylococcus mastitis. We will use in vitro co-culture systems and metagenomic methods to describe bacterial factors influencing colonization and infection of mammary glands.   Virginia Tech. Members: Christina Petersson-Wolfe In the coming year, we will work to disseminate the new findings regarding pain mitigation and the use of dexamethasone as well as to plan out our next steps for a follow-up study to continue to address this concern in the industry. We will begin to evaluate a new antimicrobial for lactating cow usage and evaluate not only the efficacy but also the associated economics. As part of our programming, we also will serve the industry through outreach to bring the science discovered at the university to the field. Hands-on workshops and seminars are planned.

Publications

Peer-Reviewed Literature   Michigan State University De Jong, E., K. D. McCubbin, D. F. Kelton, J. Sanchez, S. Dufour, S. McDougall, P.L. Ruegg, A. Lago, V. Kromker, S. M. Godden, T. J.G.M, Lam, P. J. Rajala-Schultz, J. R. Middleton, K. Orsel, J. P. Kastelic, and H. W. Barkema. 2022.  Selective treatment of non-severe clinical mastitis does not adversely affect cure, somatic cell count, milk yield, recurrence and culling:  a systematic review and meta-analysis. Available online:  <a href="https://doi.org/10.3168/jds.2022-22271">https://doi.org/10.3168/jds.2022-22271</a>   Rodriguez, Z., Q. Kolar, K. Krogstad, T. Swartz, I.Yoon,  B. Bradford and P. L. Ruegg.  Evaluation of reticuloruminal temperature for the prediction of clinical mastitis in dairy cows challenged with Streptococcus uberis. In press, J Dairy Sci, <a href="https://doi.org/10.3168/jds.2022-22421">https://doi.org/10.3168/jds.2022-22421</a>    deCampos, J.L., J.M. Strickland, J. Gandy, C. Robison, and P.L. Ruegg. 2022.  A pilot randomized clinical trial evaluating clinical effects of pegbovigrastim administered to dairy cows at dry-off. submitted Am. J. Veterinary Research. Online early publication:  <a href="https://doi.org/10.2460/ajvr.22.08.0130">https://doi.org/10.2460/ajvr.22.08.0130</a>   Gonçalves, J.L., J. Leite de Campos, A. Steinberger, N. Safdar, A. Sethi, J. Shutske, G. Suen, T. Goldberg, Roger I. Cue and P. L. Ruegg. 2022.  Incidence and treatments of mastitis and other selected bovine diseases on 37 large Wisconsin dairy farms. Pathogens 11:1282. <a href="https://doi.org/10.3390/pathogens11111282">https://doi.org/10.3390/pathogens11111282</a>   Pegolo, S., R. Tessari, V. Bisutti, D. Giannuzzi, M. Gianesella, A. Lisuzzo, E. Fiore, A. Barberio, E. Schizvon, E. Trevisi, F. P. Capelli, L. Gallo, P. Ruegg, R. Negrini, and A. Cecchinato. 2022.  Quarter-level analyses of the associations between subclinical intramammary infection and milk quality, udder health and cheese-making traits  University of Minnesota  Brink, A.A., W. J. Weber, J. D. Lippolis, J. B. Cole, S. M. Godden, A. Seykora and B. A. Crooker. 2022. Effect of Holstein genotype on ex-vivo cytokine response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during the periparturient period. Vet. Immunol. Immunopathol. <a href="https://doi.org/10.1016/j.vetimm.2022.110463">https://doi.org/10.1016/j.vetimm.2022.110463</a> Lippolis, J. D., E. J. Putz, T. A. Reinhardt, E. Casas, W. J. Weber and B. A. Crooker. 2022. Effect of Holstein genotype on immune response to an intramammary Escherichia coli challenge. J. Dairy Sci. 2021-21166. <a href="https://doi.org/10.3168/jds.2021-21166">https://doi.org/10.3168/jds.2021-21166</a>   Dean CJ, Peña-Mosca F, Ray T, Heins BJ, Machado VS, Pinedo PJ, Caixeta LS, Noyes NR. 2022. Evaluation of Contamination in Milk Samples Pooled From Independently Collected Quarters Within a Laboratory Setting. Front Vet Sci. 2022 Jun 16;9:818778. <a href="https://doi.org/10.3389/fvets.2022.818778">https://doi.org/10.3389/fvets.2022.818778</a> Ray, T., T. Gaire, C. Dean, S.M. Rowe, S.M. Godden, N.R. Noyes. 2022. The microbiome of common bedding materials before and after use on commercial dairy farms. Animal Microbiome (2022) 4: 18 <a href="https://doi.org/10.1186/s42523-022-00171-2">https://doi.org/10.1186/s42523-022-00171-2</a>   University of Missouri  Adkins PRF, Placheta L, Borchers M, Bewley J, Middleton JR.  2022.  Distribution of staphylococcal and mammaliicoccal species from compost-bedded pack or sand-bedded freestall dairy farms.  J Dairy Sci. 105(7):6261-6270.  [Epub ahead of print 13 May 22].  <a href="https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdoi.org%2F10.3168%2Fjds.2021-21500&data=05%7C01%7Cadkinsp%40missouri.edu%7C25f4c085307d4624e7c608dad706afae%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C638058720960078916%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=0z8j7JzExfSQdG44nz2OiLTAiLhcBeKUMlYGpsqfz2w%3D&reserved=0">https://doi.org/10.3168/jds.2021-21500</a> McCubbin K, de Jong E, Lam T, Kelton D, Middleton JR, McDougall S, De Vliegher S, Godden S, Rajala-Schultz P, Rowe S, Speksnijder D, Kastelic J, Barkema, H.  2022.  Invited Review: Selective use of antimicrobials in dairy cattle at drying off.  J Dairy Sci.  105(9):7161-7189.  [Epub ahead of print 2 Aug 2022].  <a href="https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdoi.org%2F10.3168%2Fjds.2021-21455&data=05%7C01%7Cadkinsp%40missouri.edu%7C25f4c085307d4624e7c608dad706afae%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C638058720960078916%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=xZBuCcN2Wv8gwzgEvNTdUsn%2Fe3KW8W2OzBL8b%2FSWaZA%3D&reserved=0">https://doi.org/10.3168/jds.2021-21455</a> Kurban D, Roy, JP, Kabera F, Frechette A, Um MM, Albaaj A, Rowe S, Godden S, Adkins PRF, Middleton JR, Gauthier ML, Keefe GP, DeVries TJ, Kelton DF, Moroni P, dos Santos MV, Barkema HW, Dufour S.  2022.  Diagnosing Intramammary Infection: Meta-analysis and Mapping Review on Frequency and Udder Health Relevance of Microorganism Species Isolated from Bovine Milk Samples.  Animals.  12(23):3288.  <a href="https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdoi.org%2F10.3390%2Fani12233288&data=05%7C01%7Cadkinsp%40missouri.edu%7C25f4c085307d4624e7c608dad706afae%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C638058720960078916%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=jIlZTq5nz%2FEkdWhsgn96pyKoeSd1tfF0Ia%2BraWy94ts%3D&reserved=0">https://doi.org/10.3390/ani12233288</a> Loy JD, Clawson ML, Adkins PRF, Middleton JR.  2022.  Current and Emerging Diagnostic Approaches to Bacterial Pathogens of Ruminants.  Vet Clin North Am Food Anim Pract.  VFP_860.  Invited Article.  In Press   Cornell University  Larsen J, Raisen CL, Ba X, Sadgrove NJ, Padilla-González GF, Simmonds MSJ, Loncaric I, Kerschner H, Apfalter P, Hartl R, Deplano A, Vandendriessche S,Černá Bolfíková B, Hulva P, Arendrup MC, Hare RK, Barnadas C, Stegger M, Sieber RN, Skov RL, Petersen A, Angen Ø, Rasmussen SL, Espinosa-Gongora C, Aarestrup FM, Lindholm LJ, Nykäsenoja SM, Laurent F, Becker K, Walther B, Kehrenberg C, Cuny C, Layer F, Werner G, Witte W, Stamm I, Moroni P, Jørgensen HJ, de Lencastre H, Cercenado E, García-Garrote F, Börjesson S, Hæggman S, Perreten V, Teale CJ, Waller AS, Pichon B, Curran MD, Ellington MJ, Welch JJ, Peacock SJ, Seilly DJ, Morgan FJE, Parkhill J, Hadjirin NF, Lindsay JA, Holden MTG, Edwards GF, Foster G, Paterson GK, Didelot X, Holmes MA, Harrison EM, Larsen AR. Emergence of methicillin resistance predates the clinical use of antibiotics. Nature. 2022 Feb;602(7895):135-141. doi: 10.1038/s41586-021-04265-w. Epub 2022, Jan 5. PMID: 34987223; PMCID: PMC8810379. Riva F, Latorre AA, Moroni P. Editorial: Ruminant mastitis: A 360° view. Front Vet Sci. 2022 Oct 27;9:1055323. doi: 10.3389/fvets.2022.1055323. PMID: 36387386; PMCID: PMC9647156. Preine F, Herrera D, Scherpenzeel C, Kalmus P, McCoy F, Smulski S, Rajala-Schultz P, Schmenger A, Moroni P, Krömker V. Different European Perspectives on the Treatment of Clinical Mastitis in Lactation. Antibiotics (Basel). 2022 Aug 16;11(8):1107. doi: 10.3390/antibiotics11081107. PMID: 36009976; PMCID:PMC9404852. Kurban D, Roy JP, Kabera F, Fréchette A, Um MM, Albaaj A, Rowe S, Godden S, Adkins PRF, Middleton JR, Gauthier ML, Keefe GP, DeVries TJ, Kelton DF, Moroni P, Veiga Dos Santos M, Barkema HW, Dufour S. Diagnosing Intramammary Infection: Meta-Analysis and Mapping Review on Frequency and Udder Health Relevance of Microorganism Species Isolated from Bovine Milk Samples. Animals (Basel). 2022 Nov 25;12(23):3288. doi: 10.3390/ani12233288. PMID: 36496808; PMCID: PMC9738497. Schell RC, Bulut E, Padda H, Safi AG, Moroni P, Ivanek R. Responsible antibiotic use labeling and consumers' willingness to buy and pay for fluid milk. J Dairy Sci. 2023 Jan;106(1):132-150. doi: 10.3168/jds.2022-21791. Epub 2022 Nov 1. PMID: 36333136. Addis MF, Pisanu S, Monistero V, Gazzola A, Penati M, Filipe J, Di Mauro S, Cremonesi P, Castiglioni B, Moroni P, Pagnozzi D, Tola S, Piccinini R. Comparative secretome analysis of Staphylococcus aureus strains with different within-herd intramammary infection prevalence. Virulence. 2022 Dec;13(1):174-190. doi: 10.1080/21505594.2021.2024014. PMID: 35030987; PMCID: PMC8765078. Dal Prà A, Biscarini F, Cavani GL, Bacchelli S, Iotti A, Borghi S, Nocetti M, Moroni P. Relationship between total and differential quarter somatic cell counts at dry-off and early lactation. PLoS One. 2022 Oct 17;17(10):e0275755. doi: 10.1371/journal.pone.0275755. PMID: 36251634; PMCID: PMC9576081. Gioia G, Freeman J, Sipka A, Santisteban C, Wieland M, Gallardo VA, Monistero V, Scott JG, Moroni P. Pathogens associated with houseflies from different areas within a New York State dairy. JDS Commun. 2022 May 21;3(4):285-290. doi:10.3168/jdsc.2021-0200. PMID: 36338025; PMCID: PMC9623797. Cremonesi P, Biscarini F, Castiglioni B, Sgoifo CA, Compiani R, Moroni P. Gut microbiome modifications over time when removing in-feed antibiotics from the prophylaxis of post-weaning diarrhea in piglets. PLoS One. 2022 Mar 7;17(3):e0262199. doi: 10.1371/journal.pone.0262199. PMID: 35255081; PMCID: PMC8901073. Casseri E, Bulut E, Llanos Soto S, Wemette M, Stout A, Greiner Safi A, Lynch R, Moroni P, Ivanek R. Understanding Antibiotic Resistance as a Perceived Threat towards Dairy Cattle through Beliefs and Practices: A Survey-Based Study of Dairy Farmers. Antibiotics (Basel). 2022 Jul 25;11(8):997. doi: 10.3390/antibiotics11080997. PMID: 35892387; PMCID: PMC9330383.   Ohio State University L.R. Larsen, L.E. Moraes, and B.D. Enger. 2022. Characteristics of mammary secretions collected from infected and uninfected primigravid dairy heifer mammary glands. J. Dairy Sci. 105:7615–7622. doi.org/10.3168/jds.2022-21794.  University of Tennessee Gelgie, A. E., Korsa, M. G. and Kerro Dego, O. 2022. Mycoplasma bovis Mastitis. Current Research in Microbial Sciences, 3, 100123. <a href="https://doi-org.utk.idm.oclc.org/10.1016/j.crmicr.2022.100123">doi:10.1016/j.crmicr.2022.100123</a>. Gelalcha, B. D.and Kerro Dego, O. 2022. Extended-Spectrum Beta-Lactamases Producing Enterobacteriaceae in the USA Dairy Cattle Farms and Implications for Public Health, Antibiotics, volume 11, issue 10,13113, doi:<a href="https://doi.org/10.3390/antibiotics11101313">10.3390/antibiotics11101313</a>. Gelalcha, B. D., Brown, S., Crocker, H., Agga, G. E. and Kerro Dego, O. 2022. Regulation Mechanisms of Virulence Genes in Enterohemorrhagic Escherichia coli. Foodborne Pathogens and Disease 19(9):598 -612. doi:<a href="https://doi.org/10.1089/fpd.2021.0103">10.1089/fpd.2021.0103</a>.   Gelalcha, B. D., Ensermu, D. B., Agga, G. E., Vancuren, M., Gillespie, B. E., D'Souza, D. H., Okafor, C. C., Kerro Dego, O. 2022. Prevalence of Antimicrobial Resistant and Extended-Spectrum Beta-Lactamase-producing Escherichia coli in Dairy Cattle Farms in East Tennessee. Foodborne Pathog Dis., 19(6), doi:<a href="https://doi.org/10.1089/fpd.2021.0101">10.1089/fpd.2021.0101</a>.   Vidlund, J., B. D. Gelalcha, S. Swanson, I. Fahrenholz, C. Deason, C. Downes and O. Kerro Dego. 2022. Pathogenesis, Diagnosis, Control, and Prevention of Bovine Staphylococcal Mastitis. In. IntechOpen. doi: 10.5772/intechopen.101596. Available at <a href="https://doi.org/10.5772/intechopen.101596">https://doi.org/10.5772/intechopen.101596</a>.  University of Vermont  Luyi Jiang, Huizeng Sun, Fengfei Gu, Jin He, Fengqi Zhao, and Jianxin Liu. (2022). Blood neutrophil extracellular traps: A novel target for the assessment of mammary health in transition dairy cows. Journal of Animal Science and Biotechnology 13:131. Doi: 10.1186/s40104-022-00782-4   Lu-Yi Jiang, Hui-Zeng Sun, Ruo-Wei Guan, Fushan Shi, Feng-Qi Zhao, Jian-Xin Liu. (2022). Formation of blood neutrophil extracellular traps increases the mastitis risk of dairy cows during the transition period. Frontiers in Immunology, in press   Fengfei Gu, L. Y. Jiang, Diming Wang, Feng-Qi Zhao, Jianxin Liu. (2022). Supplementation with N-carbamoylglutamate during the transition period improves the functions of neutrophils and reduces inflammation and oxidative stress in dairy cows. Journal of Dairy Science. doi: 10.3168/jds.2021-21159 Neher DA, Andrews TD, Weicht TR, Hurd Ad, Barlow JW 2022. Organic Farm Bedded Pack System Microbiomes: A Case Study with Comparisons to Similar and Different Bedded Packs. Dairy 2022, 3, 587–607. DOI: 10.3390/dairy3030042   Virginia Tech. Swartz, T.H. and C. S. Petersson-Wolfe. Associations between preweaning calf feeding behaviors with age at first calving and lactational performance using an automatic calf feeder. J Dairy Sci. Comm. Dec 2022 -0255. doi: 10.3168/jdsc.2022-0255. Online ahead of print.   Swartz TH, Bryant DM, Schramm HH, Duncan AJ, White RR, Wood CM, Petersson-Wolfe CS. The effects of dexamethasone administration on physiological, behavioral, and production parameters in dairy cows after a difficult calving. J Dairy Sci. 2022 Nov 15:S0022-0302(22)00663-4. doi: 10.3168/jds.2022-22029. Online ahead of print.   Liebe DM, Steele NM, Petersson-Wolfe CS, De Vries A, White RR. Practical challenges and potential approaches to predicting low-incidence diseases on farm using individual cow data: A clinical mastitis example. J Dairy Sci. 2022 Mar;105(3):2369-2379. doi: 10.3168/jds.2021-20306. Epub 2022 Jan 25. PMID: 35086707 Abstracts   Michigan State University Rodriguez, Z., Q.K. Kolar, K. Krogstad, I. Yoon, B. Bradford, and P.L. Ruegg.  2022.   Evaluation of the p<a href="https://www.mdpi.com/1424-8220/22/1/199">erformance of an intra-reticuloruminal remote sensor for the prediction of clinical mastitis </a>in dairy cows challenged with Streptococcus uberis. J Dairy Sci 105 supp 1:1408. Kolar, Q.K., K. Krogstad, Z. Rodriguez, V. Mavangira, T. Swartz, I. Yoon, B. Bradford, and P.L. Ruegg. 2022.   Effect of supplementation with Saccharomyces cerevisiae fermentation product on udder health and milk yield after intramammary challenge with S. uberis.  J Dairy Sci 105 supp 1:2288W. Kolar, Q.K., S.M. Godden, S.M., and P.L. Ruegg.  2022.  Outcomes of Treatment of Gram-positive Cases of Non-Severe Clinical Mastitis by Pathogen Group. Pp 246-247 in Proc. 61st Ann. Meeting NMC, San Diego, CA, Feb 1-3, 2022. Goncalves, J.L., J. Leite de Campos, A. Steinberger, N. Safdar, A. Sethi, J. Shutske, Z. Rodriguez, G. Suen, T. Goldberg, and P.L. Ruegg.  2022.  Outcomes of first cases of selected bovine diseases on 37 large WI dairy farms. Pp 212-213 in Proc. 61st Ann. Meeting NMC, San Diego, CA, Feb 1-3, 2022. Babiak, A.M., Q.K. Kolar, J. Leite de Campos, and P.L. Ruegg. 2022.  Accuracy of On-farm PCR Method Used for Identification of Selected Mastitis Pathogens. Pp 202-203 in Proc. 61st Ann. Meeting NMC, San Diego, CA, Feb 1-3, 2022.   University of Minnesota Lippolis, J. D., E. J. Putz, T. A. Reinhardt, E. Casas, W. J. Weber, B. A. Crooker. 2022. Holstein genotype impacts immune response to an intramammary Escherichia coli challenge. Proc. 61st Annu. Conf. National Mastitis Council. February 3. San Diego, CA. virtual. NMC Proceedings Library. <a href="http://nmconline.omnibooksonline.com/">http://nmconline.omnibooksonline.com/</a>   Lippolis, J. D., E. J. Putz, T. A. Reinhardt, E. Casas, W. J. Weber, B. A. Crooker. 2022. Whole blood transcript profiles in Holstein cows subjected to an intramammary Escherichia coli challenge. Proc. 61st Annu. Conf. National Mastitis Council. February 3. San Diego, CA. virtual. NMC Proceedings Library. <a href="http://nmconline.omnibooksonline.com/">http://nmconline.omnibooksonline.com/</a>   Pena Mosca, F., Dean, C.J., Caixeta, L.S., Ray, T., Heins, B., Machado, V.S., Pinedo, P.J., Noyes, N.R. “Description of the intramammary infections dynamics in early lactation heifer on organic dairy farms.” 54th Annual Conference of the American Association of Bovine Practitioners. Salt Lake City, UT, October 2021. Caixeta, L.S., Dow, S., Noyes, N.R., Crooker, B.A., Godden, S.M., Nydam, D.V., Walcheck, B., Wheat, W. “Effect of mucosal immune stimulant on mammary gland immune responses during dry and lactating periods in dairy cows.” Conference of Research Workers in Animal Diseases. Chicago, IL, December 2021. Dean, C. Pena-Mosca, F., Fernandes, L., Sharpe, K., Manriquez, D., Doster, E., Antunes Jr, A.M., Calles, V.F., Bauman, C., Wehri, T., Heins, B., Pinedo, P., Machado, V.S., Caixeta , L.S., Noyes, N.R., “Longitudinal survey of the bovine teat microbiome.” Conference of Research Workers in Animal Diseases. Chicago, IL, December 2021. Pena-Mosca, F., Dean, C., Ray, T., Heins, B., Pinedo, P., Machado, V.S., Caixeta, L.S., Noyes, N.R. “Association between bacterial group and persistence in the mammary gland in early lactation primiparous cows.” Conference of Research Workers in Animal Diseases. Chicago, IL, December 2021. Florentino, C.C., Pena-Mosca, F., Ruch, M., Mahmoud, M.M., Tikofsky, L., Godden, S.M., Caixeta, L.S. “The effects of administration of acidogenic boluses at dry-off in udder health and milk yield in early lactation – interim results.” 61st Annual Meeting of the National Mastitis Council, San Diego, CA. February 2022. Pena-Mosca, F., Florentino, C.C., Rocha, C., Feijoo, V.C., Masic, A., Borchers, M., Asper, D., Caixeta, L.S. “Investigation of the inclusion of on-farm measurement of somatic cell count and differential cell count in algorithms to predict cows with intramammary infections at dry-off.” 61st Annual Meeting of the National Mastitis Council, San Diego, CA. February 2022. Rowe, S.M., Dziuba, M., Boyum, B., Godden, S.M., Royster, E., Caixeta, L.S. “Negatively controlled trial investigating the effect of dry cow therapy on clinical mastitis and culling.” 61st Annual Meeting of the National Mastitis Council, San Diego, CA. February 2022. Pena Mosca, F., Florentino, C.C., Miranda, J., Rial, C., Laplacette, A., Masic, A., Borchers, M., Asper, D., Caixeta, L.S. “Assessment of the association between single somatic cell count measurement, milk culture and selective dry cow therapy algorithms.” ADSA Conference. Kansas City, MO, June 2022. Florentino, C.C., Pena Mosca, F., Ruch, M., Tikofsky, L., Knauer, W., Cramer, G., Godden, S, M., Caixeta, L.S. “ Randomized clinical trial evaluating the effects of the administration of acidogenic boluses o at dry-off on udder health.” 55th Annual Conference of the American Association of Bovine Practitioners. Long Beach, Ca. September 2022. Pena Mosca, F., Florentino, C.C., Miranda, J., Rial, C., Laplacette, A., Masic, A., Borchers, M., Asper, D., Caixeta, L.S. “Assessment of the relationship between differential somatic cell count and presence of subclinical mastitis.” 55th Annual Conference of the American Association of Bovine Practitioners. Long Beach, Ca. September 2022. Pena Mosca, F., Florentino, C.C., Rial, C., Laplacette, A., Masic, A., Borchers, M., Asper, D., Caixeta, L.S. “Investigation of the relationship between differential somatic cell count and milk culture.” 55th Annual Conference of the American Association of Bovine Practitioners. Long Beach, Ca. September 2022 Godden, S., E. Royster, E. Leonard*, D. Albrecht, J. Timmerman. Pilot Study: Refining a Culture-Guided Selective Dry Cow Therapy Program to Enhance Antimicrobial Stewardship on Dairy Farms. 55th Annu Mtg. AABP. Long Beach, CA, Sept 22-24, 2022.   Peña Mosca, F.*, S. Godden, E. Royster, D. Albrecht, B.A. Crooker, N. Aulik. Investigation of the Relationship between Manure Processing Method and Presence of M. avium subsp. paratuberculosis and Salmonella spp. in Recycled Manure Solids Bedding on Midwest Dairy Farms. 55th Annu Mtg. AABP. Long Beach, CA, Sept 22-24, 2022.   Godden, S., F. Peña Mosca, E. Royster, D. Albrecht, B.A. Crooker. Investigation of the Relationship between Manure Processing Method and Levels of Mastitis Pathogens in Recycled Manure Solids Bedding on Midwest Dairy Farms. 55th Annu Mtg. AABP. Long Beach, CA, Sept 22-24, 2022.   University of Missouri Haw SR, Adkins PRF, Middleton JR. Characterization of intramammary infections in Jersey cows. Proceedings of the 61st Annual Meeting of the National Mastitis Council, San Diego CA, Feb 1-3, 2022.   Kurban D, Roy JP, Kabera F, Frechette A, Um MM, Albaaj A, Rowe S, Godden S, Adkins PRF, Middleton JR, Gauthier ML, Keefe G, DeVries TJ, Kelton DF Moroni P, dos Santos MV, Barkema HW, and Dufour S.  2022.  Diagnosing intramammary infections: meta-analysis and mapping review on frequency and udder health relevance of microorganism species retrieved in bovine milk samples. Proceedings of the 61st Annual Meeting of the National Mastitis Council, San Diego CA, Feb 1-3, 2022.   Haw S, Adkins PRF, Middleton JR, Bernier Gosselin V.  Pathogen-specific intramammary infection prevalence, persistence, and somatic cell count association in lactating Jersey cows.  Proceedings of the 44th Annual CVM Research Day.  6 May 2022.  Abstract #52   Isensee P and Adkins P. Characterization of the antibacterial activity of Staphylococcus chromogenes isolates originating from dairy cattle. MU CVM Research Day. May 6, 2022   Kurban D, Roy JP, Kabera F, Frechette A, Um MM, Albaaj A, Rowe S, Godden S, Adkins PRF, Middleton JR, Gauthier ML, Keefe G, DeVries TJ, Kelton DF Moroni P, dos Santos MV, Barkema HW, and Dufour S.  2022.  Diagnosing intramammary infection: Meta-analysis and mapping review on frequency and udder-health relevance of microorganism species retrieved from bovine milk samples.  16th International Symposium of Veterinary Epidemiology and Economics, Halifax Nova Scotia, Canada.  August 7-12, 2022.   Ohio State University   Baker, P.H., K.M. Enger, S.K. Jacobi, R.M. Akers, B.D. Enger. 2022. Effect of Staphylococcus aureus intramammary infection on heifer mammary gland growth and development. J. Dairy Sci. 105 (Suppl. 1):177. Baker, P.H., K.M. Enger, S.K. Jacobi, R.M. Akers, B.D. Enger. 2022. Cellular proliferation in Staphylococcus aureus infected heifer mammary glands that were hormonally stimulated to rapidly grow. J. Dairy Sci. 105 (Suppl. 1):301. Larsen, L.R., P.H. Baker, K.M. Enger, L.E. Moraes, and B.D. Enger. 2022. Prepartum administration of internal teat sealant in primigravid dairy heifers to reduce intramammary infection prevalence at calving. Pages 272-273 in Natl. Mastitis Counc. Annu. Mtg. Proc., San Diego, CA, Natl. Mastitis Council Inc., New Prague, MN. Baker, P.H., F.K. Arnold, D.D. Clevenger, S.K. Jacobi, R.M Akers, and B.D. Enger. 2022. Histological response in hormonally stimulated mammary glands of non-pregnant dairy heifers to a Staphylococcus aureus intramammary infection. Pages 228-229 in Natl. Mastitis Counc. Annu. Mtg. Proc., San Diego, CA, Natl. Mastitis Council Inc., New Prague, MN.  Oregon State University Ford HR, Zheng T, Bionaz M, Huo Q, Hasan D. 2022. The D2Dx immunity test as a measure of immune health in ewes and lambs. J. Dairy Sci. Vol. 105:163 Suppl. 1 Ford HR, Bionaz M, Trevisi E, Hasan D. 2022. Feeding chicory silage to transition ewes has a minimal effect on blood metabolic parameters but a strong effect on blood micromineral levels. J. Dairy Sci. Vol. 105:157 Suppl. 1 Ford HR, Bionaz M, Ates S, Trevisi E. 2022. A combination of chicory-plantain silage and Se-yeast have a minimal effect on blood biomarkers during intramammary infection in lactating ewes. J. Dairy Sci. Vol. 105:109 Suppl. 1   University of Vermont   Heins K, Chakrawarti A, Eckstrom K, Mugabi R, Barlow JW 2022 Genetic Variation in The Type I Restriction-Modification Systems of Staphylococcus aureus from Selected Vermont Dairy Farm and their Association with Antimicrobial-Resistant Genes. ASM Microbe, American Society of Microbiology Joint Annual Meeting, June 9-13, Washington, DC. Prandini SC, Jeffrey CE, Chakrawarti A, Barlow JW 2022. Case Report: Hock Lesions as a Source of Staphylococcus aureus Mastitis. Proceedings of National Mastitis Council, 61st Annual Meeting, San Diego CA. Astmann TJ, Jeffrey CE, Chakrawarti A, Barlow JW 2022. Bacteria Isolated from Bovine Teat Skin Inhibit Staphylococcus aureus in an in vitro Simultaneous Antagonist Assay. Proceedings of National Mastitis Council, 61st Annual Meeting, San Diego CA. Chakrawarti A, Eckstrom K, Laaguiby P, Mugabi R, Burke A, Barlow JW 2021. Concordances between whole genome sequencing and conventional methods for pathogen strain typing and antimicrobial resistance of Staphylococcus aureus isolated from humans, cattle and milk on cheese making dairy farms. World Microbe Forum, American Society of Microbiology Joint   Annual Meeting, June 20-24, virtual.   Conference Proceedings   Michigan State University Ruegg, P.L. 2022. Microbiology, Maldi-Tof, Microbiome and more:  how to understand and use diagnostic tests to improve udder health. In Proc. Am Assoc Bov Pract   Ruegg, P.L. 2022. Treatment of clinical mastitis. What we know and where we go. Pp 62-71 in Proc. 61st Ann. Meeting NMC, San Diego, CA, Feb 1-3, 2022. University of Missouri Adkins PRF. Molecular Diagnostics – The Good, The Bad, and The Ugly. Proceedings of the 61st Annual Meeting of the National Mastitis Council, San Diego CA, Feb 1-3, 2022. University of Tennessee Desai, S., Vidlund, J. and Kerro Dego, O. 2022. Evaluation of protective efficacy of novel staphylococcal mastitis vaccines in cattle. The 26th Annual Exhibition of Undergraduate Research and Creative Achievement (EURēCA) Symposium, April 25, The University of Tennessee, Knoxville. Gelgie, A. E., Gillespie, B. E., Almeida, R., Lysnyansky, I., Shpigel, N. Y., Agga, G. E. and Kerro Dego, O. 2023. Identification of virulence genes of Mycoplasma bovis by transposon mutation and experimental infection of dairy cows. Accepted for presentation at Conference of Research Workers on Animal Disease (CRWAD), January 20 -24, Chicago, IL.   Poster Presentations   Outreach Presentations   University of Minnesota     Caixeta, L.S. “Behind a successful lactation is a proper dry-off.” <ul> <li>November 2021: Western Dairy Management Conference, Reno, NV</li> <li>March 2022: Central Plains Dairy Expo, Sioux Falls, SD</li> </ul>