All-trans retinoic acid is increased in the acute phase-related hyporetinemia during Escherichia coli mastitis

Blood vitamin A profiles, including concentrations of retinol and its active metabolite retinoic acid, were assessed during the peripartum period and during experimentally induced Escherichia coli mastitis in heifers. Serum retinol decreased in all animals in the immediate postpartum period and normalized within 1 wk after parturition. No significant changes were detectable in the concentrations of retinoic acid isomers during puerperium. Following intramammary E. coli infusion, all cows showed moderate symptoms of systemic disease besides the local signs of inflammation. The presence of a systemic acute-phase reaction was documented by fever, increase in serum amyloid A, and decrease in serum albumin. Retinol concentration in serum also decreased spectacularly during coliform mastitis, and the decline was clearly related to the timing of the acute-phase response. Moreover, a significant increase of all-trans retinoic acid, mirrored by a lowering of 13-cis retinoic acid, was detected during the same time period. The 9-cis isomer of retinoic acid was present in all samples, but it remained below the quantification limit. Results confirmed the decrease in serum retinol during the peripartum period of dairy cows. Furthermore, the study established that profound changes in vitamin A metabolism occur during the acute-phase reaction of coliform mastitis in heifers. The bovine infection model reproduced the acute phase-related hyporetinemia, as previously observed in humans and rats. In addition, all-trans retinoic acid was found to be the most abundant circulating acid isomer during mastitis, providing an indication for a possible key role of all-trans retinoic acid in the modulation of the immune response.     

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 μg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.     

Immunobiology of hematopoietic colony-stimulating factors: potential application to disease prevention in the bovine.

Colony-stimulating factors are a family of glycoproteins instrumental in regulation of hematopoiesis and inflammation. Clinical effects of various colony-stimulating factors have been reported in murine and human hosts. This review summarizes findings from some clinical trial evaluations of macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-6, and interleukin-7 administration to other species. These factors stimulate clonal expansion of progenitor cells in the bone marrow, induce differentiation of various cell lineages to a mature phenotype, and, in some cases, enhance the effector activities of immune cells. Each colony-stimulating factor has distinct lineages of bone marrow cells upon which they act, although there is some overlap in lineage activity and synergy between colony-stimulating factors. The close relationship in biological activity among different colony-stimulating factors is also reflected at the genomic level at which genes for some hematopoietic growth factors have been mapped to a region of human chromosome 5. Recently, colony-stimulating factor administration to cattle and its potential application to disease control in bovine preventive medicine programs has been investigated. Data from recent hematological, immunological, and intramammary bacterial (Staphylococcus aureus and Klebsiella pneumoniae) challenge studies in dairy cows are reviewed. These studies, with limited numbers of cows, found that rate of new infections, as well as duration and severity of infection, were reduced by pretreatment of cows with granulocyte-colony stimulating factor. The dose-dependent hematological and immunomodulatory effects of granulocyte colony-stimulating factor administration may explain reduced severity and incidence of mastitis in dairy cows given granulocyte colony-stimulating factor.     

A longitudinal observational study of the dynamics of Mycoplasma bovis antibodies in naturally exposed and diseased dairy cows

Mycoplasma bovis is an important pathogen causing disease and substantial economic losses in cattle. However, knowledge of the dynamics of antibody responses in individual cows in the face of an outbreak is currently extremely limited. The use of commercial antibody tests to support clinical decision-making and for surveillance purposes is therefore challenging. Our objective was to describe the dynamics of M. bovis antibody responses in 4 Danish dairy herds experiencing an acute outbreak of M. bovis-associated disease, and to compare the antibody dynamics between dairy cows with different disease manifestations. A total of 120 cows were examined using a standardized clinical protocol and categorized into 4 disease groups: “mastitis,” “systemic,” “nonspecific,” and “none.” Paired blood and milk samples were collected and tested using a commercial M. bovis antibody–detecting ELISA. Plots of raw data and generalized additive mixed models with cow and herd as random effects were used to describe serum and milk antibody dynamics relative to the estimated time of onset of clinical disease. Cows with mastitis had high optical density measurement (ODC%) of antibodies in both milk and serum at disease onset. The estimated mean ODC% in milk was below the manufacturer's cut-off for the other groups for the entire study period. The estimated mean serum ODC% in the “systemic” group was high at onset of disease and stayed above the cut-off until 65 d after disease onset. However, the lower 95% confidence interval (CI) for the mean ODC% was only above the manufacturer's cut-off between 7 and 17 d after onset of disease. The CI of the “systemic” and “none” groups did not overlap at any time between the day of disease onset and 65 d after disease onset, and the estimated mean ODC% for both the “nonspecific” and “none” groups were generally below the cut-off for the majority of the study period. In conclusion, the serum antibody responses were highly dynamic and showed a high level of variation between individual cows. This strongly suggests that serology is unlikely to be useful for individual diagnosis of M. bovis-associated disease in dairy cows. However, it might still be useful for herd- or group-level diagnosis. Antibodies in milk were only increased in cows with M. bovis mastitis, indicating that milk antibody measurements only have diagnostic utility for cows with mastitis.     

Metabotypes with elevated protein and lipid catabolism and inflammation precede clinical mastitis in prepartal transition dairy cows

Clinical mastitis (CM), the most prevalent and costly disease in dairy cows, is diagnosed most commonly shortly after calving. Current indicators do not satisfactorily predict CM. This study aimed to develop a robust and comprehensive mass spectrometry-based metabolomic and lipidomic workflow using untargeted ultra-performance liquid chromatography high-resolution mass spectrometry for predictive biomarker detection. Using a nested case-control design, we measured weekly during the prepartal transition period differences in serum metabolites, lipids, inflammation markers, and minerals between clinically healthy Holstein dairy cows diagnosed with mastitis postcalving (CMP; n = 8; CM diagnosis d 1 = 3 cows, d 2 = 2 cows, d 4 = 1 cow; d 25 = 1 cow, and d 43 = 1 cow that had subclinical mastitis since d 3) or not (control; n = 9). The largest fold differences between CMP and control cows during the prepartal transition period were observed for 3′-sialyllactose in serum. Seven metabolites (N-methylethanolamine phosphate, choline, phosphorylcholine, free carnitine, trimethyl lysine, tyrosine, and proline) and 3 metabolite groups (carnitines, AA metabolites, and water-soluble phospholipid metabolites) could correctly classify cows for their future CM status at both 21 and 14 d before calving. Biochemical analysis using lipid and metabolite-specific commercial diagnostic kits supported our mass spectrometry-based omics results and additionally showed elevated inflammatory markers (serum amyloid A and visfatin) in CMP cows. In conclusion, metabolic phenotypes (i.e., metabotype) with elevated protein and lipid metabolism and inflammation may precede CM in prepartal transition dairy cows. The discovered serum metabolites and lipids may assist in predictive diagnostics, prevention strategies, and early treatment intervention against CM, and thereby improve cow health and welfare.     

Reduced levels of total leukocytes and neutrophils in Norwegian cattle selected for decreased mastitis incidence

Two groups of Norwegian Cattle showing significant genetic differences in clinical mastitis susceptibility were examined for hematological changes at three stages of lactation. Blood samples were taken from 91 healthy Norwegian Cattle cows and heifers belonging either to a high protein yield group or to a low clinical mastitis group and analyzed for hematological properties and serum cortisol levels at three stages of lactation. All animals were free of intramammary infections at the time of sampling. All cows had increased total white blood cells, neutrophils, and neutrophil/lymphocyte ratio as parturition approached, with peak levels at parturition. Cows selected for low clinical mastitis had lower levels of total white blood cells and neutrophils compared with cows selected for high protein yield throughout all three periods. The difference was significant prepartum. Cows from the low clinical mastitis group also had lower neutrophil/lymphocyte ratios and lower serum cortisol levels than those in the high protein yield group. A significant positive correlation was found between cortisol and total white blood cells and neutrophils, respectively. We conclude that selection for lower mastitis incidence in the low clinical mastitis group is associated with a significant decrease in total white blood cells and neutrophils in blood prepartum. Results from the present study, and the genetic difference in mastitis incidence observed in the groups, indicates that increased leukocyte mobilization at certain stages of lactation may be associated with increased susceptibility to mastitis.     

Milk and serum IL-4, IL-6, IL-10, and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci

The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).     

Association between serum calcium dynamics around parturition and common postpartum diseases in dairy cows

The objective of this study was to compare periparturient serum Ca dynamics (CaDyn) in cows with and without diseases in early lactation. The study enrolled 1,949 cows from a commercial dairy farm in northern Germany. Blood samples were drawn 7 d before expected calving date and on d 0, 1, 3, and 7 after calving and analyzed for serum Ca concentration. Cows were monitored for clinical hypocalcemia (CH), ketosis, left displaced abomasum (LDA), retained placenta, acute puerperal metritis (APM), mastitis, and pneumonia. To evaluate the association between CaDyn and diseases during the transition period, repeated measures ANOVA with first-order autoregressive covariance were performed. Serum CaDyn of healthy cows (i.e., without any of the aforementioned diseases) was compared with CaDyn of cows with one of the aforementioned diseases (CH, ketosis, APM, mastitis, LDA, and pneumonia), and cows with multiple diseases (CH+, ketosis+, APM+, mastitis+, LDA+, and pneumonia+). Separate models were built for primiparous and multiparous cows. For primiparous cows, we evaluated the association between CaDyn and ketosis (healthy cows vs. cows with ketosis vs. cows with ketosis+) and CaDyn and APM (healthy cows vs. cows with APM vs. cows with APM+). The same models were built for multiparous cows. Three additional models were built for multiparous cows to evaluate the association between CaDyn and CH (healthy cows vs. cows with CH vs. cows with CH+), mastitis (healthy cows vs. cows with mastitis vs. cows with mastitis+), or LDA (healthy cows vs. cows with LDA vs. cows with LDA+). In primiparous cows, serum Ca concentrations of cows with ketosis, APM, and APM+ were significantly reduced on d 3 and 7 after calving, compared with healthy cows. Serum Ca concentrations of primiparous cows with ketosis+ were reduced on d 3, but not on d 7 after calving. Multiparous cows with CH had significantly reduced serum Ca concentrations on d 0, 1, and 3 compared with healthy cows. On d 3 and 7, serum Ca concentration of CH+ cows was significantly reduced compared with healthy multiparous cows. Multiparous cows with ketosis and ketosis+ had significantly reduced serum Ca concentrations on d 1 and 3 compared with healthy cows. Cows with APM+ had significantly increased serum Ca concentrations on d 0 and reduced serum Ca concentrations on d 3, compared with healthy cows. Whereas multiparous cows with mastitis had a reduced serum Ca concentration on d 1, mastitis+ cows had a reduced serum Ca concentration on d 1 and 3, compared with healthy multiparous cows. Overall, multiparous cows with LDA+ had reduced serum Ca concentrations. Especially a delayed onset of hypocalcemia (d 3 and 7) was indicative for the development of disease in primiparous cows. In multiparous cows, reduced serum Ca concentrations on d 1 and 3 were associated with occurrence of diseases. Future studies should evaluate whether reduced serum Ca concentrations are a cause or concomitant circumstance of diseases in early lactation.     

Association of CXCR2 polymorphisms with subclinical and clinical mastitis in dairy cattle

The ability to identify objectively cows that are more or less susceptible to mastitis has been a long-standing goal. Genetic markers associated with inflammatory responses during mastitis could aid in selection of these cattle. One potential marker is CXCR2, a chemokine receptor required for neutrophil migration to infection sites, which contains single nucleotide polymorphisms (SNP) within the gene. The objective of this experiment was to evaluate the association of CXCR2 SNP genotypes with subclinical and clinical mastitis. Thirty-seven Holstein and 42 Jersey cows that completed at least 2 full lactations were used. Quarter foremilk samples were collected for bacteriological examination quarterly and when cows exhibited clinical mastitis. Subclinical mastitis was defined as the presence of the same pathogen in the same quarter in at least 2 of 3 consecutive samples. A significant association was detected between CXCR2 SNP +777 genotype and percentages of subclinical mastitis cases in Holsteins. Holsteins expressing genotype GG had decreased percentages of subclinical mastitis, but genotype CC cows had increased percentages of subclinical mastitis. Significant differences in clinical mastitis incidence were not detected between genotypes for either breed. This approach of genetically identifying mastitis resistant cows may represent an effective means of marker-assisted selection for mastitis and other inflammatory diseases involving neutrophils.     

High cortisol concentrations and mediation of the hypogalactia during endotoxin-induced mastitis.

A series of experiments was conducted to define the role of cortisol in the hypogalactia during endotoxin-induced mastitis. In the first experiment, three of six nonmastitic cows were given a continuous infusion of trilostane, a 3 beta-hydroxysteroid dehydrogenase inhibitor that blocks enhanced cortisol synthesis. Trilostane had no effect in these cows. In the second experiment, six midlactation cows were given 10 micrograms of endotoxin in each of two homolateral quarters to induce mastitis. Three of these cows also received trilostane. Increased serum cortisol following endotoxin infusion was blocked by trilostane treatment, whereas serum glucose and rectal temperatures were unaffected. Preventing the cortisol increase failed to reduce hypogalactia in endotoxin-infused or uninfused quarters. Decreases in milk production and increases in measures of mammary inflammation were greater in trilostane-treated cows, indicating that endogenous cortisol may moderate the cow's inflammatory response. In the third experiment, three of six nonmastitic cows were injected intramuscularly with 150 IU of ACTH. Serum cortisol concentration exceeded 70 ng/ml for at least 3 h in cows receiving ACTH. This cortisol concentration, comparable with concentrations found during endotoxin mastitis, did not inhibit milk production. Together, these data demonstrate that the acute cortisol increase does not mediate the hypogalactia associated with endotoxin-induced mastitis.     

Effect of clinical mastitis on the lactation curve: a mixed model estimation using daily milk weights

The objective of this study was to estimate the milk production losses associated with clinical mastitis using mixed linear models and correlation structures that have not been available previously. Data used included computer-recorded daily milk yields and detailed and accurate recordings of clinical mastitis cases. Two commercial Holstein dairy farms in New York State participated in the study, one with 650 lactating cows and another that began the study with 830 lactating cows and increased to 1120 cows by the end of the study. Cows on both farms were housed in free stall barns and milked 3 times daily in milking parlors. Electrical conductivity was used as a diagnostic aid for clinical mastitis on both farms. Date of clinical onset was recorded for every episode of clinical mastitis as well as for 8 other diseases defined using standardized case definitions (dystocia, milk fever, retained placenta, metritis, ketosis, displaced abomasum, lameness, and cystic ovarian disease) during the study period of October 1, 1999 to July 31, 2001. The mixed linear model for explaining variation in the outcome variable daily milk yield relative to non-mastitic herdmates found the terms for all 9 diseases studied, including clinical mastitis, significant. The model with an autoregressive correlation structure was preferred based on -2 * log likelihood, Akaike's information criterion, and Bayesian information criterion as well as savings in degrees of freedom. Separate analyses were run for first lactation cows and for second-plus lactation cows because their lactation curves were shaped differently. Adjusting for the effects of the other 8 diseases, milk production loss from clinical mastitis during the whole lactation was estimated as approximately 598 kg for second-plus lactation cows. However, cows that contracted mastitis had a daily production advantage of 2.6 kg over their herdmates until they contracted the disease. When compared with this potentially higher milk production, the total loss from clinical mastitis was estimated as 1181 kg.     

Effect of dexamethasone on experimental Escherichia coli mastitis in the cow

The effect of intramuscular administration of dexamethasone on milk production and on several clinical and hematologic indicators in cows with experimental Escherichia coli mastitis was studied. Two groups of three cows each were intramammarily inoculated with 20 x 10(5) cfu E. coli 0101 K99 F41 in rear quarters. Immediately after inoculation one groups received 30 mg dexamethasone intramuscularly, whereas the other group was injected similarly with pyrogen-free saline. Fourteen days later milk production losses were markedly less in the dexamethasone-treated group. Dexamethasone treatment reduced local clinical signs of inflammation, significantly increased rectal temperatures, and diminished inhibition of rumen amplitude during the acute phase of infection. Moreover, cows treated with dexamethasone showed diminished neutropenia, more pronounced hypozincemia, and delayed onset in the decrease in plasma iron concentrations. Possible mechanisms by which these changes are accomplished are discussed.     

Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction

Nonpregnant, midlactation primiparous Holstein cows were fed ad libitum (n = 12) or at 80% of maintenance energy requirements (n = 12) to determine whether feed restriction influences clinical response to endotoxin-induced mastitis. After 2 wk of ad libitum or restricted feeding, one mammary quarter per cow was infused with 100 microg of endotoxin. Within 3 to 6 h of intramammary infusion, endotoxin increased mean rectal temperature, heart rate, and milk somatic cell count and immunoglobulin (IgG) concentration; and decreased blood leukocyte count and rumen motility in both restricted and ad libitum-fed cows. Mean serum and milk tumor necrosis factor-alpha (TNF-alpha) concentrations showed only modest increases following endotoxin infusion. Restricted fed cows had slightly different acute fever responses and significantly increased heart and respiration rates than ad libitum fed cows. However, feed restriction did not influence mean total leukocyte count, rumen motility, serum TNF-a concentrations or milk IgG and TNF-alpha concentrations. Thus, results of this study suggest that energy balance does not significantly alter clinical symptoms following acute endotoxin-induced mastitis, at least in midlactation cows. As such, negative energy balance may not underlie the increases in severe coliform mastitis commonly observed in periparturient dairy cows.     

Short communication: Association of disease incidence and adaptive immune response in Holstein dairy cows

The objective of this study was to use previously calculated estimated breeding values for cell- (CMIR) and antibody-mediated immune responses (AMIR) to determine associations between immune response (IR) and economically important diseases of dairy cattle. In total, 699 Holsteins were classified as high, average, or low for CMIR, AMIR, and overall IR (combined CMIR and AMIR), and associations with mastitis, metritis, ketosis, displaced abomasums, and retained fetal membranes were determined. The incidence of mastitis was higher among average cows as compared with cows classified as high AMIR [odds ratio (OR)=2.5], high CMIR (OR=1.8), or high IR (OR=1.8). Low-CMIR cows had a higher incidence of metritis (OR=11.3) and low-IR cows had a higher incidence of displaced abomasum (OR=4.1) and retained fetal membrane (OR=2.8) than did average responders. Results of this study show that cows classified as high immune responders have lower occurrence of disease, suggesting that breeding cattle for enhanced IR may be a feasible approach to decrease the incidence of infectious and metabolic diseases in the dairy industry.     

Symposium review: Oxylipids and the regulation of bovine mammary inflammatory responses

Inflammation is a critical aspect of the innate immune system that can determine the outcome of several economically important diseases of dairy cattle such as mastitis. The purpose of the inflammatory response is to eliminate the source of tissue injury and then return tissues to normal function. Aggressive or uncontrolled inflammatory responses, however, can damage host tissues and contribute significantly to the pathophysiology associated with mastitis. A precarious balance between pro-inflammatory and pro-resolving mechanisms is needed to ensure optimal pathogen clearance and a prompt return to immune homeostasis. Therefore, inflammatory responses must be tightly regulated to avoid bystander damage to the milk-synthesizing tissues of the mammary gland. Oxylipids are potent lipid mediators that can regulate all aspects of the inflammatory response. The biosynthetic profiles of oxylipids are dependent on both the availability of diverse polyunsaturated fatty acids substrates and their subsequent metabolism through various oxidizing pathways. Changes in lipid metabolism in dairy cows around parturition can profoundly change the composition and concentration of oxylipids in the mammary gland that may be responsible for dysfunctional inflammatory responses during this time. This review will provide a brief overview of the bovine inflammatory response and the role that oxylipids play in contributing to the onset and resolution of inflammation especially as it pertains to mastitis. Factors associated with periparturient cows that can contribute to dysfunctional regulation of inflammation as a function of altered oxylipid biosynthesis and metabolism also will be described. Understanding the role that oxylipids may play in the development of mastitis is key to developing novel prevention and control programs for the dairy industry.     

Evaluation of breed-dependent differences in the innate immune responses of Holstein and Jersey cows to Staphylococcus aureus intramammary infection

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection with Staphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-gamma, interleukin (IL)-12, tumour growth factor(TGF)-alpha, and TGF-beta1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows to Staph. aureus intramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.     

Natural variation in biomarkers indicating mastitis in healthy cows

Dairy herds are expanding and, with increasing numbers of animals in each herd, there is a need for automatic recording of indicators in milk in order to detect mastitis, inflammation of the udder. A number of biomarkers for mastitis have been suggested over the years. Mastitis usually occurs in one of the four udder quarters and since it is now possible to milk each udder quarter separately in automated milking systems, it is important to evaluate the normal variation in the biomarkers at udder quarter level. This study evaluated the normal variations between milkings for some biomarkers in clinically healthy cows, determined by repeated somatic cell count and bacteriological analysis. The biomarkers studied were serum amyloid A (SAA), haptoglobin (Hp), lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAGase) and alkaline phosphatase (AP), parameters that have been suggested as markers for mastitis. Ten cows were monitored on 42 consecutive milking occasions through collection of udder quarter milk samples and representative cow composite milk samples, giving a total of 2100 individual milk samples. Each cow had its individual profile for the concentrations and variations in the parameters analysed. Although there was relatively large variation between cows for the biomarkers analysed, the variation between milkings in clinically healthy quarters within cows was often below 10%. The biomarker with the lowest variation in this study was LDH. The results suggest that comparing quarters within an individual cow can identify deviations from the natural variations between milkings. This could be a valuable tool instead of, or in combination with, a cut-off value for each parameter in order to detect changes in the milk indicating mastitis.     

Short communication: Diagnosis and classification of clinical and subclinical mastitis utilizing a dynamometer and a handheld infrared thermometer

In times of ongoing automatization of dairy cow husbandry, objective and reliable tools for mastitis diagnostic are highly in demand. The objective of this study was to investigate the diagnostic value of a handheld dynamometer and an infrared thermometer to diagnose and score clinical and subclinical mastitis and to compare those values with results from palpation of the udder tissue. Overall, 218 cows with clinical mastitis (i.e., 46 mild, 106 moderate, and 66 severe cases), 142 with subclinical mastitis, and 68 healthy cows were enrolled. Our data provide evidence that the dynamometer is an accurate diagnostic tool to differentiate between healthy udder quarters, and those with subclinical and clinical mastitis. Furthermore, the severity score of clinical mastitis can be estimated by dynamometer. The firmness threshold for the detection of clinical mastitis was 1.002 kg. Using a threshold of 1.175 kg in clinical mastitis quarters, it was possible to differentiate between negative and positive bacteriological results. A differentiation between healthy and clinical mastitis quarters with the infrared thermometer was possible, albeit udder surface temperatures were highly influenced by ambient temperature. Udder surface temperature increased by 0.15 to 0.18°C for each degree of ambient temperature. In conclusion, the utility of an infrared thermometer to estimate the udder health status of dairy cows is limited, whereas the handheld dynamometer appeared to be an accurate and objective method.     

Short communication: Oxidative status and incidence of mastitis relative to blood α-tocopherol concentrations in the postpartum period in dairy cows

Vitamin E supplementation, when combined with high blood α-tocopherol (>6.25 μg/mL) at dry off, has been reported to unexpectedly increased the risk for clinical mastitis in dairy cows. Furthermore, higher levels of oxidative stress in the postpartum period were related to higher risk of mastitis. The objective of the present study was to determine the relationship between various serum biomarkers of oxidative status, incidence of mastitis, and blood α-tocopherol concentrations at dry off and at calving. A total of 146 dairy cows from a commercial farm were used in an observational field study. All cows were supplemented with 3,000 and 50 IU/cow per day of all-rac-α-tocopherol during the dry period and lactation, respectively. Blood samples were collected at dry off and at calving. Serum was analyzed for α-tocopherol, levels of reactive oxygen metabolites (ROM), thiol groups (SH), and ferric-reducing ability. Three α-tocopherol groups at calving were created: high (>3 μg/mL), medium (2–3 μg/mL), and low (<2 μg/mL). Three α-tocopherol groups at dry off were created: high (>6.25 μg/mL), medium (4.25–6.25 μg/mL), and low (<4.25 μg/mL). All cases of clinical mastitis that occurred during the dry period and the entire subsequent lactation were verified by a veterinarian. No differences were observed in the incidence of mastitis between the 3 α-tocopherol groups based on the serum levels at dry off. Incidence of mastitis was 4 times lower in the high and medium groups when compared with the corresponding value for the low-α-tocopherol group based on the serum levels at calving. Lower levels of ROM and SH at dry off and at calving were found in the group of cows with the highest α-tocopherol values at dry off when compared with the corresponding values in the low-α-tocopherol group. The ROM values at dry off but not at calving were lower in the group of cows with the highest α-tocopherol values at calving when compared with the corresponding values in the low-α-tocopherol group. No differences were observed in ferric-reducing ability values between the 3 α-tocopherol groups at dry off or calving. No differences were observed in all biomarkers of oxidative status between healthy cows and those with mastitis. Thus, blood α-tocopherol is inversely related to certain biomarkers of oxidative stress in the postpartum period and incidence of mastitis. However, reduction in the incidence of mastitis is not mediated through a reduction in the levels of various biomarkers of oxidative stress.     

Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity

Bovine mastitis is mainly caused by bacterial infection and is responsible for important economic losses as well as alterations of the health and welfare of animals. The increase in somatic cell count (SCC) in milk during mastitis is mainly due to the influx of neutrophils, which have a crucial role in the elimination of pathogens. For a long time, these first-line defenders have been viewed as microbe killers, with a limited role in the orchestration of the immune response. However, their role is more complex: we recently characterized a bovine neutrophil subset expressing major histocompatibility complex class II (MHC-II) molecules (MHC-II^pos), usually distributed on antigen-presenting cells, as having regulatory capacities in cattle. In this study, our objective was to evaluate the implication of different neutrophils subsets in the mammary gland immunity during clinical and subclinical mastitis. Using flow cytometry, we analyzed the presence of MHC-II^pos neutrophils in blood and in milk during clinical mastitis at different time points of inflammation (n = 10 infected quarters) and during subclinical mastitis, defined as the presence of bacteria and an SCC >150,000 cells/mL (n = 27 infected quarters). Our results show, for the first time, that in blood and milk, neutrophils are a heterogeneous population and encompass at least 2 subsets distinguishable by their expression of MHC-II. In milk without mastitis, we observed higher production of reactive oxygen species and higher phagocytosis capacity of MHC-II^pos neutrophils compared with their MHC-II^neg counterparts, indicating the high bactericidal capacities of MHC-II^pos neutrophils. MHC-II^pos neutrophils are enriched in milk compared with blood during subclinical mastitis but not during clinical mastitis. Moreover, we observed a positive and highly significant correlation between MHC-II^pos neutrophils and T lymphocytes present in milk during subclinical mastitis. Our experiments involved a total of 47 cows (40 Holstein and 7 Normande cows). To conclude, our study opens the way to the discovery of new biomarkers of mastitis inflammation.