Effect of Holstein genotype on immune response to an intramammary Escherichia coli challenge.

Selective breeding of US dairy cows since the mid-1960s has contributed to remarkable gains in milk yield per cow. This increased milk yield has been associated with an increase in health issues. Since 1964, the University of Minnesota has selectively bred a Holstein herd to maintain genetically static, unselected Holsteins (UH). Comparison of these UH cows with contemporary Holsteins (CH) has demonstrated that the UH cows not only produce less milk but also have fewer health concerns than their CH herdmates. The objective of this study was to determine the effects of Holstein genotype on innate immune response in an experimental intramammary Escherichia coli challenge model. Primiparous UH (n = 5) and CH (n = 7) cows received 430 cfu of E. coli strain P4 in 1 quarter. Blood and affected quarter milk samples were collected at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 7, 9, and 11 d relative to E. coli infusion. Rectal temperatures were recorded at each milking through d 4 of the experiment. Milk bacterial counts, somatic cell count and BSA concentrations, complete blood cell counts, rectal temperature, and serum and milk whey cytokine (IL-1ß and IL-6) concentrations were used as metrics to determine infection severity. Longitudinal (repeated) 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 CH than UH cows from d 0.25 through d 2.5. Milk somatic cell count increased within 6 h (d 0.25) after E. coli administration in CH and UH cows but did not differ between genotypes after d 1. Rectal body temperature peaked at d 1 in CH and UH cows but was greater in CH cows. Milk BSA, IL-1ß, and IL-6 concentrations were greater in CH than UH cows after E. coli administration. Blood lymphocyte and neutrophil counts were decreased at 0.5 and 1 d in CH but not in UH cows. The number of differentially expressed transcripts at each of the postinfusion sampling times was consistently greater (4- to 90-fold) in CH than in UH 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 UH cows but became more systemic in the CH cows. These data demonstrate that UH cows 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 CH cows with an immune system that is less effective in fighting intramammary infections. Identification of genetic factors associated with enhanced immune functions that differ between the UH and CH cows could contribute to efforts to reintroduce or enhance beneficial components that have been lost or reduced in the CH cows since the mid-1960s.

Bovine NK-lysin-derived peptides have bactericidal effects against Mycobacterium avium subspecies paratuberculosis.

Infection with Mycobacterium avium subspecies paratuberculosis (MAP) is complex, but little is known about the role that natural killer (NK) cells play. In the present study, four bovine NK-lysin peptides were synthesized to evaluate their bactericidal activity against MAP. The results demonstrated that bNK-lysin peptides were directly bactericidal against MAP, with bNK1 and bNK2A being more potent than bNK2B and bNK2C. Mechanistically, transmission electron microscopy revealed that the incubation of MAP with bNK2A resulted in extensive damage to cell membranes and cytosolic content leakage. Furthermore, the addition of bNK2A linked with a cell-penetrating peptide resulted in increased MAP killing in a macrophage model.

Case report: characterization of a persistent, treatment-resistant, novel Staphylococcus aureus infection causing chronic mastitis in a Holstein dairy cow.

BACKGROUND: Mastitis is the most common health concern plaguing the modern dairy cow and costs dairy producers estimates of two billion dollars annually. Staphylococcus aureus infections are prevalent, displaying varied disease presentation and markedly low cure rates. Neutrophils are considered the first line of defense against mastitis causing bacteria and are frequently targeted in the development of treatment and prevention technologies. We describe a case of naturally occurring, chronic mastitis in a Holstein cow (1428), caused by a novel strain of S. aureus that was not able to be cleared by antibiotic treatment. CASE PRESENTATION: The infection was identified in a single quarter, 2 months into the cow's first lactation. The infection persisted for the following 20 months, including through dry off, and a second calving and lactation. This case of mastitis was associated with a consistently high somatic cell count, however presented with no other clinical signs. This cow was unsuccessfully treated with antibiotics commonly used to treat mastitis, consisting of two rounds of treatment during lactation and an additional round at the beginning of dry off. The chronic infection was also unchanged through an experimental mid-lactation treatment with pegylated granulocyte-colony stimulating factor (PEG-gCSF) and an additional periparturient treatment with PEG-gCSF. We isolated milk neutrophils from 1428 and compared them to two cows challenged with experimental S. aureus, strain Newbould 305. Neutrophils from 1428's milk had higher surface expression of myeloperoxidase compared to experimental Newbould challenged animals, as well as increased presence of Neutrophil Extracellular Traps. This suggests a heightened activation state of neutrophils sourced from 1428's naturally occurring infection. Upon postmortem examination, the affected quarter revealed multifocal abscesses separated by fibrous connective tissues. Abscesses were most common in the gland cistern and collecting duct region. Microscopically, the inflammatory reaction was pyogranulomatous to granulomatous and consistent with botryomycosis. Colonies of Gram-positive cocci were found within the eosinophilic matrix of the Splendore-Hoeppli reaction within granulomas and intracellularly within the acinar epithelium. CONCLUSIONS: Collectively, we describe a unique case of chronic mastitis, the characterization of which provides valuable insight into the mechanics of S. aureus treatment resistance and immune escape.

Dataset of bovine mammary gland dry secretion proteome from the end of lactation through day 21 of the dry period.

This dataset is a label-free quantitation of proteins milk and dry secretions from the end of lactation through day 21 of the dry period using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The data supplied in this article supports the accompanying publication entitled "Characterization of bovine mammary gland dry secretions and their proteome from the end of lactation through day 21 of the dry period" [1]. The Thermo mass spectrometry raw files and MaxQuant files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset number PXD017837.

Characterization of bovine mammary gland dry secretions and their proteome from the end of lactation through day 21 of the dry period.

The dry period in dairy cows is critical to mammary health. We used label-free mass spectroscopy to quantify whey proteins from milk/dry secretions collected on days 0, 3,10, and 21 of the dry period. The number of proteins identified was 776. The number of proteins upregulated at one or more times in the dry period was 109. The most significant enriched Gene Ontology (GO) terms for the upregulated proteins were immune function and stress-related. The number of proteins that are downregulated in the dry period was 68. The most significant enriched GO terms for the down-regulated proteins were stress and immune function related. In other assays, total protein and lactoferrin concentrations rose significantly by days 10 and 21 of the dry period. We then examined day 21 dry secretions for their ability to inhibit bacterial growth. Bacterial growth inhibition did not correlate with lactoferrin for 4 coliforms tested nor for all 7 mastitis pathogens tested. Eleven proteins out of the total number of proteins identified were significantly associated with positive or negative coliform growth in day 21 dry secretions. The dry secretion protein composition changes we found may provide new information for understanding the dry period and indirectly involution. BIOLOGICAL SIGNIFICANCE: The dry period in a cow is critical to the success of a cow's subsequent lactation for many reasons. Of particular importance is the rate of establishment of new intramammary infections (IMI), which is significant in the dry period. IMI susceptibility is most significant in the first few weeks of the dry period. There are both physical and mechanical reasons for increased IMI susceptibility in the first few weeks of the dry period. We also know that dry secretions have high levels of antimicrobial proteins and bacterial growth is diminished in dry secretions compared to milk. However, we found that by day 21 of the dry period, antimicrobial proteins, such as lactoferrin, are no longer correlated with reduced bacterial growth in dry secretions. We established a dry secretion proteome for the first 3 weeks of the dry period. The library of proteins in dry secretions presented provides a better understanding of the critical dry period in dairy cow management.

Genome Sequence of a Staphylococcus aureus Strain Isolated from a Dairy Cow That Was Nonresponsive to Antibiotic Treatment.

Staphylococcus aureus can cause mastitis in dairy cattle. We report the genome sequence of a Staphylococcus aureus strain isolated from a dairy cow with a chronic case of mastitis. The infection with this strain of Staphylococcus aureus was not cleared from the animal with antibiotic treatment.

Lactation stage impacts the glycolytic function of bovine CD4+ T cells during ex vivo activation.

Dairy cattle undergo dynamic physiological changes over the course of a full lactation into the dry period, which impacts their immunocompetence. During activation, T cells undergo a characteristic rewiring to increase the uptake of glucose and metabolically reprogram to favor aerobic glycolysis over oxidative phosphorylation. To date it remains to be completely elucidated how the altered energetic demands associated with lactation in dairy cows impacts T cell metabolic reprogramming. Thus, in our ex vivo studies we have examined the influence of stage of lactation (early lactation into the dry period) on cellular metabolism in activated bovine CD4+ T cells. Results showed higher rates of glycolytic function in activated CD4+ T cells from late lactation and dry cows compared to cells from early and mid-lactation cows. Similarly, protein and mRNA expression of cytokines were higher in CD4+ T cells from dry cows than CD4+ T cells from lactating cows. The data suggest CD4+ T cells from lactating cows have an altered metabolic responsiveness that could impact the immunocompetence of these animals, particularly those in early lactation, and increase their susceptibility to infection.

MicroRNA profiles of dry secretions through the first three weeks of the dry period from Holstein cows.

In dairy cows, the period from the end of lactation through the dry period and into the transition period, requires vast physiological and immunological changes critical to mammary health. The dry period is important to the success of the next lactation and intramammary infections during the dry period will adversely alter mammary function, health and milk production for the subsequent lactation. MicroRNAs (miRNAs) are small non-coding RNAs that can post transcriptionally regulate gene expression. We sought to characterize the miRNA profile in dry secretions from the last day of lactation to 3, 10, and 21 days post dry-off. We identified 816 known and 80 novel miRNAs. We found 46 miRNAs whose expression significantly changed (q-value < 0.05) over the first three weeks of dry-off. Additionally, we examined the slopes of random regression models of log transformed normalized counts and cross analyzed the 46 significantly upregulated and downregulated miRNAs. These miRNAs were found to be associated with important components of pregnancy, lactation, as well as inflammation and disease. Detailing the miRNA profile of dry secretions through the dry-off period provides insight into the biology at work, possible means of regulation, components of resistance and/or susceptibility, and outlets for targeted therapy development.

Carbon monoxide exposures among U.S. wildland firefighters by work, fire, and environmental characteristics and conditions.

Carbon monoxide (CO) exposure levels encountered by wildland firefighters (WLFs) throughout their work shift can change considerably within a few minutes due to the varied tasks that are performed and the changing environmental and fire conditions encountered throughout the day. In a U.S. Forest Service study during the 2009-2012 fire seasons, WLFs from 57 different fires across the U.S. were monitored for CO using CO data-logging detectors while an observer recorded worker tasks, fire characteristics, and environmental conditions at scheduled intervals. Exposures to CO for 735 WLF's work shifts were analyzed to assess the effect of variations among work tasks, fire characteristics, and environmental conditions. Geometric mean full shift time-weighted averages were low at 2.4 parts per million (ppm) and average length of work shift was 11 hr and 15 min. The task with the highest mean CO exposure was sawyer/swamper at 6.8 ppm; workers performing that task had an estimated 9 times higher odds of a having a 1-min CO measurement exceeding 25 ppm than the referent pump task (OR = 8.89, 95% CI = 1.97, 40.24). After adjusting CO exposure limits for shift length, elevation, and work level, 2% and 4% of the WLF's work shifts exceeded the National Institute for Occupational Safety and Health's recommended exposure level and the American Conference of Governmental Industrial Hygienist's threshold limit value, respectively. In regression modeling, variables that were significantly associated with elevated levels of CO exposure included: task, fuel model, wind orientation, crew type, relative humidity, type of attack, and wind speed. In the absence of instruments such as CO detectors that can determine and alert WLFs to elevated CO levels, recognition of the conditions that lead to elevated levels of CO exposure can assist WLFs to effectively use administrative controls, such as work rotations, to minimize exposures.

Synthetic bovine NK-lysin-derived peptide (bNK2A) does not require intra-chain disulfide bonds for bactericidal activity.

Bovine NK-lysins are cationic antimicrobial proteins found predominantly in the cytosolic granules of T lymphocytes and NK-cells. NK-lysin-derived peptides show antimicrobial activity against both Gram positive and Gram negative bacteria. Mature NK-lysin protein has six well-conserved cysteine residues. This study was performed to assess whether synthetic bovine NK-lysin-derived peptide (bNK2A) forms disulfide bonds and whether disulfide bonds were essential for bNK2A antimicrobial activity. Two 30-mer bNK2A peptides were synthesized: one with two original cysteines and an analog with cysteines substituted with two serines. Mass spectrometry revealed lack of disulfide bonds in original peptide while CD spectrophotometry showed both peptides have similar α-helical structures. Since both peptides were equally inhibitory to Histophilus somni, disulfide bonds appeared dispensable for synthetic bNK2A peptide antibacterial activity.

Wildland firefighter smoke exposure and risk of lung cancer and cardiovascular disease mortality.

Wildland firefighters are exposed to wood smoke, which contains hazardous air pollutants, by suppressing thousands of wildfires across the U. S. each year. We estimated the relative risk of lung cancer and cardiovascular disease mortality from existing PM2.5 exposure-response relationships using measured PM4 concentrations from smoke and breathing rates from wildland firefighter field studies across different exposure scenarios. To estimate the relative risk of lung cancer (LC) and cardiovascular disease (CVD) mortality from exposure to PM2.5 from smoke, we used an existing exposure-response (ER) relationship. We estimated the daily dose of wildfire smoke PM2.5 from measured concentrations of PM4, estimated wildland firefighter breathing rates, daily shift duration (hours per day) and frequency of exposure (fire days per year and career duration). Firefighters who worked 49 days per year were exposed to a daily dose of PM4 that ranged from 0.15 mg to 0.74 mg for a 5- and 25-year career, respectively. The daily dose for firefighters working 98 days per year of PM4 ranged from 0.30 mg to 1.49 mg. Across all exposure scenarios (49 and 98 fire days per year) and career durations (5-25 years), we estimated that wildland firefighters were at an increased risk of LC (8 percent to 43 percent) and CVD (16 percent to 30 percent) mortality. This unique approach assessed long term health risks for wildland firefighters and demonstrated that wildland firefighters have an increased risk of lung cancer and cardiovascular disease mortality.

Milk biosynthesis requires the Golgi cation exchanger TMEM165.

Milk is a hallmark of mammals that is critical for normal growth and development of offspring. During biosynthesis of lactose in the Golgi complex, H+ is produced as a by-product, and there is no known mechanism for maintaining luminal pH within the physiological range. Here, using conditional, tissue-specific knockout mice, immunostaining, and biochemical assays, we test whether the putative H+/Ca2+/Mn2+ exchanger known as TMEM165 (transmembrane protein 165) participates in normal milk production. We find TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and for normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased osmosis-mediated dilution of the milk caused by the decreased biosynthesis of lactose. When normalized to total protein levels, only calcium and manganese levels were significantly lower in the milk from TMEM165-deficient dams than control dams. These findings suggest that TMEM165 supplies Ca2+ and Mn2+ to the Golgi complex in exchange for H+ to sustain the functions of lactose synthase and potentially other glycosyl-transferases. Our findings highlight the importance of cation and pH homeostasis in the Golgi complex of professional secretory cells and the critical role of TMEM165 in this process.

Factors affecting smoke and crystalline silica exposure among wildland firefighters.

Smoke exposure data among U.S. wildland firefighters for carbon monoxide, respirable particulate and respirable crystalline silica are presented from a field surveillance program between 2009 and 2012. Models to predict fireline-average exposure to each inhalation hazard were developed and fit to the available data. The models identify factors to consider when defining similar exposure groups and designing future data collection. Task-based rather than shift-average data collection is important because the work activity representing the majority of fireline time, the position up- or downwind of the fire, and the proportion of time this combination represented were significant factors in the model for carbon monoxide, and all but wind position were significant for respirable particulate matter. The wind position versus the fire was not important for respirable quartz exposure. The crew type was an important factor in each model.

Membrane and Cytoplasmic Proteins of Mycobacterium avium subspecies paratuberculosis that Bind to Novel Monoclonal Antibodies.

Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis (Map) proteins are important tools in Johne's disease research and diagnostics. Johne's disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of Map, the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding Map antigens for 14 mAbs that could not be identified previously. In the first approach, a new Map-lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to Map proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes.

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis.

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality.

Avian Intestinal Mucus Modulates Campylobacter jejuni Gene Expression in a Host-Specific Manner.

Campylobacter jejuni is a leading cause of bacterial foodborne illness in humans worldwide. However, C. jejuni naturally colonizes poultry without causing pathology where it resides deep within mucus of the cecal crypts. Mucus may modulate the pathogenicity of C. jejuni in a species-specific manner, where it is pathogenic in humans and asymptomatic in poultry. Little is known about how intestinal mucus from different host species affects C. jejuni gene expression. In this study we characterized the growth and transcriptome of C. jejuni NCTC11168 cultured in defined media supplemented with or without mucus isolated from avian (chicken or turkey) or mammalian (cow, pig, or sheep) sources. C. jejuni showed substantially improved growth over defined media, with mucus from all species, showing that intestinal mucus was an energy source for C. jejuni. Seventy-three genes were differentially expressed when C. jejuni was cultured in avian vs. mammalian mucus. Genes associated with iron acquisition and resistance to oxidative stress were significantly increased in avian mucus. Many of the differentially expressed genes were flanked by differentially expressed antisense RNA asRNA, suggesting a role in gene regulation. This study highlights the interactions between C. jejuni and host mucus and the impact on gene expression, growth and invasion of host cells, suggesting important responses to environmental cues that facilitate intestinal colonization. IMPORTANCE  Campylobacter jejuni infection of humans is an important health problem world-wide and is the leading bacterial cause of foodborne illnesses in U.S. The main route for exposure for humans is consumption of poultry meat contaminated during processing. C. jejuni is frequently found in poultry, residing within the mucus of the intestinal tract without causing disease. It is not clear why C. jejuni causes disease in some animals and humans, while leaving birds without symptoms. To understand its activity in birds, we characterized C. jejuni responses to poultry mucus to identify genes turned on in the intestinal tract of birds. We identified genes important for colonization and persistence within the poultry gut, turned on when C. jejuni was exposed to poultry mucus. Our findings are an important step in understanding how C. jejuni responds and interacts in the poultry gut, and may identify ways to reduce C. jejuni in birds.

Genomic and Transcriptomic Analysis of Escherichia coli Strains Associated with Persistent and Transient Bovine Mastitis and the Role of Colanic Acid.

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. It is most often transient in nature, causing an infection that lasts 2 to 3 days. However, E. coli has been shown to cause a persistent infection in a minority of cases. Mechanisms that allow for a persistent E. coli infection are not fully understood. The goal of this work was to determine differences between E. coli strains originally isolated from dairy cattle with transient and persistent mastitis. Using RNA sequencing, we show gene expression differences in nearly 200 genes when bacteria from the two clinical phenotypes are compared. We sequenced the genomes of the E. coli strains and report genes unique to the two phenotypes. Differences in the wca operon, which encodes colanic acid, were identified by DNA as well as RNA sequencing and differentiated the two phenotypes. Previous work demonstrated that E. coli strains that cause persistent infections were more motile than those that cause transient infections. Deletion of genes in the wca operon from a persistent-infection strain resulted in a reduction of motility as measured in swimming and swarming assays. Furthermore, colanic acid has been shown to protect bacteria from complement-mediated killing. We show that transient-infection E. coli strains were more sensitive to complement-mediated killing. The deletion of genes from the wca operon caused a persistent-infection E. coli strain to become sensitive to complement-mediated killing. This work identifies important differences between E. coli strains that cause persistent and transient mammary infections in dairy cattle.

Genome Sequences of Escherichia coli Strains That Cause Persistent and Transient Mastitis.

We report here the genome sequences of two strains of Escherichia coli (ECA-B and ECC-M) that cause bovine mastitis. These strains are known to be associated with persistent and transient mastitis; strain ECA-B causes a transient infection, and ECC-M leads to a persistent infection.

Differential Gene Expression of Three Mastitis-Causing Escherichia coli Strains Grown under Planktonic, Swimming, and Swarming Culture Conditions.

Bacterial motility is thought to play an important role in virulence. We have previously shown that proficient bacterial swimming and swarming in vitro is correlated with the persistent intramammary infection phenotype observed in cattle. However, little is known about the gene regulation differences important for different motility phenotypes in Escherichia coli. In this work, three E. coli strains that cause persistent bovine mastitis infections were grown in three media that promote different types of motility (planktonic, swimming, and swarming). Using whole-transcriptome RNA sequencing, we identified a total of 935 genes (~21% of the total genome) that were differentially expressed in comparisons of the various motility-promoting conditions. We found that approximately 7% of the differentially expressed genes were associated with iron regulation. We show that motility assays using iron or iron chelators confirmed the importance of iron regulation to the observed motility phenotypes. Because of the observation that E. coli strains that cause persistent infections are more motile, we contend that better understanding of the genes that are differentially expressed due to the type of motility will yield important information about how bacteria can become established within a host. Elucidating the mechanisms that regulate bacterial motility may provide new approaches in the development of intervention strategies as well as facilitate the discovery of novel diagnostics and therapeutics. IMPORTANCE Bacteria can exhibit various types of motility. It is known that different types of motilities can be associated with virulence. In this work, we compare gene expression levels in bacteria that were grown under conditions that promoted three different types of E. coli motility. Better understanding of the mechanisms of how bacteria can cause an infection is an important first step to better diagnostics and therapeutics.

Vitamin D status of dairy cattle: Outcomes of current practices in the dairy industry.

The need for vitamin D supplementation of dairy cattle has been known for the better part of the last century and is well appreciated by dairy producers and nutritionists. Whether current recommendations and practices for supplemental vitamin D are meeting the needs of dairy cattle, however, is not well known. The vitamin D status of animals is reliably indicated by the concentration of the 25-hydroxyvitamin D [25(OH)D] metabolite in serum or plasma, with a concentration of 30ng/mL proposed as a lower threshold for sufficiency. The objective of this study was to determine the typical serum 25(OH)D concentrations of dairy cattle across various dairy operations. The serum 25(OH)D concentration of 702 samples collected from cows across various stages of lactation, housing systems, and locations in the United States was 68±22ng/mL (mean ± standard deviation), with the majority of samples between 40 and 100ng/mL. Most of the 12 herds surveyed supplemented cows with 30,000 to 50,000 IU of vitamin D3/d, and average serum 25(OH)D of cows at 100 to 300 DIM in each of those herds was near or above 70ng/mL regardless of season or housing. In contrast, average serum 25(OH)D of a herd supplementing with 20,000 IU/d was 42±15ng/mL, with 22% below 30ng/mL. Cows in early lactation (0 to 30d in milk) also had lower serum 25(OH)D than did mid- to late-lactation cows (57±17 vs. 71±20ng/mL, respectively). Serum 25(OH)D of yearling heifers receiving 11,000 to 12,000 IU of vitamin D3/d was near that of cows at 76±15ng/mL. Serum 25(OH)D concentrations of calves, on the other hand, was 15±11ng/mL at birth and remained near or below 15ng/mL through 1mo of age if they were fed pasteurized waste milk with little to no summer sun exposure. In contrast, serum 25(OH)D of calves fed milk replacer containing 6,600 and 11,000 IU of vitamin D2/kg of dry matter were 59±8 and 98±33ng/mL, respectively, at 1mo of age. Experimental data from calves similarly indicated that serum 25(OH)D achieved at approximately 1mo of age would increase 6 to 7ng/mL for every 1,000 IU of vitamin D3/kg of dry matter of milk replacer. In conclusion, vitamin D status of dairy cattle supplemented with vitamin D3 according to typical practices, about 1.5 to 2.5 times the National Research Council recommendation, is sufficient as defined by serum 25(OH)D concentrations. Newborn calves and calves fed milk without supplemental vitamin D3, however, are prone to deficiency.