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.     

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.     

The value of the biomarkers cathelicidin,milk amyloid A,and haptoglobin to diagnose and classify clinical and subclinical mastitis

Timely and objective diagnosis and classification of mastitis is crucial to ensure adequate management and therapeutic decisions. Analyzing specific biomarkers in milk could be advantageous compared with subjective or semiquantitative criteria, such as palpation of the udder in clinical mastitis cases or evaluation of somatic cell count using cow side tests (e.g., California Mastitis Test) in subclinical mastitis quarters. The objective of this study was to investigate the diagnostic value of 3 biomarkers; cathelicidin, milk amyloid A, and haptoglobin for the diagnosis of subclinical and clinical mastitis. Furthermore, the suitability of these biomarkers to differentiate between mild, moderate, and severe clinical mastitis and the influence of different pathogens on biomarker levels was tested. A total of 67 healthy cows, 119 cows with subclinical mastitis, and 212 cows with clinical mastitis were enrolled in the study. Although cathelicidin, haptoglobin, and milk amyloid A were measured in all samples from healthy cows and those with subclinical mastitis, haptoglobin, and cathelicidin results were only available from 121 out of 212 cows with clinical mastitis. Milk amyloid A was measured in all samples. In cows with clinical mastitis, the mastitic quarter and a second healthy quarter serving as a healthy in-cow control quarter were sampled. It was possible to differentiate between healthy quarters, quarters with subclinical mastitis, and quarters with clinical mastitis using all 3 biomarkers. Concerning cathelicidin, thresholds were 0.000 [sensitivity (Se) = 0.83, specificity (Sp) = 0.97] and 0.053 (Se = 0.98, Sp = 0.99) for normalized optical density at 450 nm (NOD450) for differentiating between healthy quarters and quarters with subclinical or clinical mastitis, respectively. Thresholds of 1.28 µg/mL (Se = 0.65, Sp = 0.76) and 1.81 µg/mL (Se = 0.77, Sp = 0.83) for milk amyloid A and 3.65 µg/mL (Se = 0.92, Sp = 0.94) and 5.40 µg/mL mL (Se = 0.96, Sp = 0.99) for haptoglobin were calculated, respectively. Healthy in-cow control quarters from cows with CM showed elevated milk amyloid A and haptoglobin levels compared with healthy quarters from healthy cows. Only the level of milk amyloid A was higher in severe clinical mastitis cases compared with mild ones. In contrast to clinical mastitis, cathelicidin and haptoglobin in subclinical mastitis quarters were significantly influenced by different bacteriological results. The measurement of cathelicidin, milk amyloid A, and haptoglobin in milk proved to be a reliable method to detect quarters with subclinical or clinical mastitis.     

Determination of milk and blood concentrations of lipopolysaccharide-binding protein in cows with naturally acquired subclinical and clinical mastitis

Blood and milk concentrations of the acute phase protein lipopolysaccharide-binding protein (LBP) were evaluated in cows with naturally occurring mastitis. Blood and milk samples were collected from 101 clinically healthy dairy cows and 17 dairy cows diagnosed with clinical mastitis, and the LBP concentrations of the samples were measured by an ELISA. Concentrations of LBP were greater in the blood and milk of cows with clinical mastitis than in those with healthy quarters. Concentrations of LBP also differed between uninfected and subclinically infected quarters with low somatic cell count. Blood concentrations of LBP in cows with subclinical intramammary infections could not be differentiated from those of cows with all healthy quarters. Together, these data demonstrate that increased blood and milk concentrations of LBP can be detected in dairy cows with naturally acquired intramammary infections that cause clinical mastitis.     

Serum concentration and mRNA expression in milk somatic cells of toll-like receptor 2, toll-like receptor 4, and cytokines in dairy cows following intramammary inoculation with Escherichia coli

The objective of the current study was to investigate the toll-like receptors (TLR), including the soluble forms sTLR2 and sTLR4, involved in innate immune responses of dairy cows to experimentally induced Escherichia coli mastitis. Six clinically healthy Holstein dairy cows received an intramammary inoculation of E. coli O111:K58 between 63 and 83 d postpartum. Concentrations of sTLR2 and sTLR4, the proinflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α), and acute phase proteins serum amyloid A (SAA) and haptoglobin (Hp) in blood were measured by ELISA. Furthermore, 10 mL of milk was collected from challenged quarters immediately before inoculation and at 6, 12, 24, 48, and 72 h after inoculation, and mRNA expression of selected genes, including TLR2, TLR4, IL-1β, IL-6, TNF-α, and IL-8, was quantified by real-time PCR. Escherichia coli intramammary infection elicited a decrease in the circulating levels of leukocytes. Rectal temperature was elevated at 6 h postinoculation (PI). Similarly, the serum concentrations of TNF-α, IL-6, and SAA increased at 6 h PI. However, serum concentrations of sTLR2, sTLR4, and Hp did not differ after challenge. The mRNA expression of TLR2, IL-1β, and IL-8 in milk somatic cells increased at 12 h PI, whereas a decreased IL-6 mRNA expression was detected from 6 to 48 h PI. In conclusion, we found that TLR2 mRNA expression increased in milk somatic cells collected from infected quarters of cows challenged with E. coli, whereas the concentrations of sTLR2 and sTLR4 remained unchanged after challenge. Thus, sTLR2 and sTLR4 may protect the host by sequestrating pathogen-associated molecular patterns during E. coli mastitis.     

Imbalance between lipoxin A4 and leukotriene B4 in chronic mastitis-affected cows

Persistent accumulation of inflammatory cells in the udder, with neutrophils being the predominant cell type, is a characteristic feature of chronic mastitis in dairy cows. Leukotriene (LT) B4 is a potent chemotactic agent, known to induce recruitment and accumulation of neutrophils in the bovine mammary gland. The LTB4-stimulated neutrophil functional responses are closely opposed by lipoxin (LX) A4, which promotes the resolution of inflammation. We thus hypothesized that the chronic inflammation of the udder could be associated with an unfavorable ratio between these two eicosanoids and that the persistence of neutrophil accumulation could be due to an increase in LTB4 synthesis and/or an impaired LXA4 production. In an attempt to verify this hypothesis, we first measured LXA4, LTB4, and their ratio in the milk of healthy and acute and chronic mastitis-affected quarters. Next, we studied the relationships between these variables and the degree of udder inflammation as assessed by somatic cell count measurement. The LTB4 concentration was low in healthy quarters, drastically increased in acute mastitis, and reached intermediate levels in chronic mastitis-affected quarters. However, whereas LXA4 concentration was highly increased in acute mastitis, healthy and chronic quarters had similarly low values. The LXA4:LTB4 ratio was thus significantly lower in chronic mastitis-affected cows. The LTB4 concentrations measured in chronic quarters were highly correlated to somatic cell count and to milk neutrophil and macrophage numbers. A weaker correlation was observed between LXA4 and these variables. For both eicosanoids, the highest correlation was observed with the number of neutrophils. These results show the existence of an LXA4:LTB4 imbalance in chronic mastitis-affected cows because of low LXA4 concentrations. Further studies are needed to determine whether administration of LX or stable analogs could have therapeutic potential in the control of chronic bovine mastitis.     

Relationship between haptoglobin and serum amyloid A in milk and milk quality

The objective of this study was to evaluate relationships between the presence in milk of the major bovine acute phase proteins, haptoglobin (Hp) and serum amyloid A (SAA), and milk quality parameters. Composite milk samples were collected from 89 clinically healthy dairy cows and analysed for Hp and SAA, total protein, casein, and whey protein levels, casein number, proteolysis, total fat and lactose levels, and somatic cell count (SCC). Milk samples with detectable levels of Hp showed lower total protein and casein levels than those samples without Hp, whereas milk samples with detectable levels of SAA had lower casein number and lactose level than samples without detectable SAA. Samples with detectable levels of acute phase proteins also showed an elevated SCC. The results suggest that the presence of Hp and SAA in milk might indicate unfavourable changes in milk composition, especially in relation to protein quality.     

Dose dependency and individual variability of the lipopolysaccharide-induced bovine acute phase protein response

In order to investigate the dose dependency and the individual variability of the lipopolysaccharide (LPS)-induced acute phase protein response in cattle, 8 nonlactating, nonpregnant Danish Holstein cows were challenged 3 times each by intravenous injection of increasing doses (10, 100, and 1000 ng/kg, consecutively) of Escherichia coli LPS with 3-wk intervals. All 3 LPS doses resulted in a rapid increase in serum concentrations of haptoglobin and serum amyloid A (SAA) and a decrease in serum concentrations of albumin in all 8 cows. Serum concentrations of acute phase proteins (APP) remained altered for several days after each LPS injection, and their increase or decrease was significantly related to LPS dose. In addition to dose dependency, the response was also dependent on the individual, as APP concentrations differed significantly among cows. To compare APP production in 2 consecutive challenges, individual APP levels after the challenge with 100 ng LPS/kg were correlated to levels attained after the challenge with 1000 ng LPS/kg. Serum amyloid A concentrations correlated between the 2 challenges, whereas haptoglobin concentrations tended to correlate; no correlation could be demonstrated between SAA and haptoglobin concentrations in either of the challenges, which suggests that the synthesis of haptoglobin and SAA are regulated in different ways. In conclusion, cattle are highly susceptible to LPS, as very low doses of LPS elicited acute phase albumin, SAA, and haptoglobin responses. Concentrations of APP not only reflect the magnitude of LPS exposure but are also influenced by the ability of the individual cow to mount an acute phase response. The ability to produce SAA and haptoglobin may be an innate characteristic of the individual, as responses in consecutive challenges were quantitatively similar.     

Prepartum milking of heifers influences future production and health

Transition heifers face multiple stressors during the periparturient period, including first exposure to milking, that may adversely impact dry matter intake (DMI), reduce milk production, compromise immune function, and increase susceptibility to disease. It was hypothesized that reducing the combined stressors experienced at calving would improve the periparturient performance, health, and well-being of heifers. The objective of this study was to determine the effect of initiating the milking procedure 3 wk before expected calving on production, DMI, body weight, energy balance, udder health, calving traits, and health status, as indicated by plasma acute phase protein concentrations. Twenty-two primigravid heifers, blocked by expected calving date, were assigned randomly either to a prepartum milking (PM) group or control group. The PM heifers were milked twice daily beginning at 21 d before expected calving, and control heifers were not milked until after calving. All heifers had access to the same precalving and post-calving diets. Results indicated that PM heifers produced more milk during the first 2 wk after calving and had greater DMI as a percentage of body weight during the first month after calving than did control heifers, although energy balance was more negative for PM heifers. The PM heifers had reduced somatic cell counts through the first month after calving and lower average somatic cell scores during lactation despite having more quarters with mastitis infection at calving. The PM heifers had less udder edema at the third milking postcalving, and had reduced concentrations of haptoglobin in blood sooner than did control heifers. These results indicate that prepartum milking is an alternative management practice that has beneficial effects on the production, health, and well-being of first-lactation cows.     

Milk L-lactate concentration is increased during mastitis

A study was undertaken in cattle to evaluate changes in milk L-lactate in relation to mastitis. A healthy, rear quarter of the udder of each of ten cows in mid-lactation was infused with 1000 colony-forming units (cfu) of Streptococcus uberis following an afternoon milking. Foremilk samples were taken at each milking from control and treated quarters and antibiotic treatment was applied following the onset of clinical mastitis or after 72 h. One cow did not become infected. Six quarters showed clinical symptoms of mastitis within 24-40 h and this was associated with a more than 30-fold increase in milk L-lactate (to 3.3 mM) and an increase in somatic cell count (SCC) from 4.5 x 10(3) to 1 x 10(7) cells/ml. Three cows were subclinical, with cell counts ranging from 1.5 x 10(6) to 1 x 10(7) cells/ml. In these animals, milk lactate ranged from 0.7 to 1.5 mM in the infected quarters up to 40 h post-infection, compared with less than 0.1 mM in control quarters. Milk was examined from 137 cows in mid-lactation which were known to have mastitis. Foremilk samples were taken aseptically from control and infected quarters of cows on commercial farms. Mean milk L-lactate concentrations and SCC were 0.14 +/- 0.02 mM and 1.85 +/- 0.3 x 10(5) cells/ml, respectively, in control (bacteriologically negative) samples. However, L-lactate concentrations exceeded 2.5 mM in the presence of some types of infection, the level of the lactate response being closely related to the impact of the infection on SCC. L-Lactate concentrations were relatively elevated in milk samples taken post partum, declining from 0.8 to 0.14 mM oyer the first few days of lactation. In conclusion, milk L-lactate has potential as an indicator of clinical and subclinical mastitis in dairy cows.     

Effect of severity of systemic signs during the acute phase of experimentally induced Escherichia coli mastitis on milk production losses

The objectives were to describe the systemic signs during the acute phase of experimental Escherichia coli mastitis and to relate these with losses in milk production during the reconvalescent period. Eleven cows, 20 to 30 d postpartum, were inoculated with 10(4) cfu of E. coli O157 in rear quarters. Heart rate, rectal temperature, frequency and amplitude of rumen contractions, plasma Zn and Fe concentrations, and counts of E. coli were used to monitor severity of disease during the acute phase. Areas under curves of clinical parameters, plasma Zn and Fe concentrations, and counts of E. coli were calculated during 36, 120, and 125 h postinoculation, respectively. Areas under curves of milk production were calculated during 21 d postinoculation. Losses in total daily milk production were related positively with areas under curves of heart rate, rumen amplitude, counts of E. coli in secreta from inoculated quarters, and plasma Zn and Fe concentrations. These parameters may prove suitable to establish an accurate prognosis for returning to milk production cows suffering from acute or peracute E. coli mastitis and to evaluate efficacy of drugs in experimental mastitis.     

Interaction of somatic cell count and quarter milk flow patterns

Milk flow parameters at udder and quarter levels were studied in relation to somatic cell count (SCC) and other risk factors for mastitis (bimodality, duration of decline, and duration of overmilking phase). Thirty-eight Holstein cows in their first to sixth lactations were investigated during 10 mo of lactation. Monthly milk samples were collected for SCC during morning milking. Quarter and udder milk flows were recorded daily. A cow was included if one quarter was found to have an SCC higher than 200 × 10³ cells/mL. A total of 3,262 quarter milk flow curves and 804 udder milk flow curves from 22 cows (6 primiparous and 16 multiparous) were selected and evaluated. Selected data for milk flow profiles in relation to SCC represented 5 consecutive morning milkings around the time of milk sampling (sampling on d 3). A total of 661 milk samples were analyzed. At both the udder and quarter levels milk yield was reduced in groups with increased SCC. Quarters with high SCC (>500 × 10³ cells/mL) had lower peak flow rate and longer overmilking phases compared with quarters with low SCC (<200 × 10³ cells/mL). There was a tendency for a longer duration of the decline phase in quarters with high SCC but no effect was observed at the udder level. There were longer declines in bimodal milk flows at the quarter, but not at the udder, level. Also, quarters with bimodality had longer overmilking phases. The duration of the decline phases at the quarter level influenced all measured parameters except the duration of the increase phase. The quarters with a longer duration of the decline phase (≥80 s) had greater SCC and peak flow rate but had lower milk yield compared with quarters with a shorter duration of the decline phase (<27 s). Duration of the overmilking phase influenced all measured parameters except SCC. We conclude that for good udder health, the duration of the decline phase at the quarter level should be considered for milking parameters and udder preparation before milking.     

Effect of mastitis on proteolytic activity in bovine milk

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.     

Relationship between somatic cell count,individual leukocyte populations and milk components in bovine udder quarter milk

The somatic cell count (SCC) in bovine milk is an indicator of udder health and milk quality. Individual cell populations, i.e., macrophages, lymphocytes and polymorphonuclear neutrophils, were identified and their relationship to milk components such as acute-phase proteins (serum amyloid A (SAA) and α₁-acid glycoprotein), lactic acid and lactoferrin (LF), as well as protein, fat and lactose, were studied in foremilk from separate udder quarters. The whey proteins and peptides were studied using two-dimensional gel electrophoresis, which was shown in the present study to be a useful method of assessing udder health and milk quality. The growth of a starter culture was evaluated using a conductance method as a simulation of fermentation processes relevant for cheese production. The investigation showed that LF, SAA, l-lactate and lactose are reliable indicators of milk quality on udder quarter level. The correlations between SCC, or the individual cell populations, and the potential indicators were highly significant.     

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cow's immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56 h. At 48 h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48 h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens.     

Role of endotoxin and TNF-alpha in the pathogenesis of experimentally induced coliform mastitis in periparturient cows

Twelve cows were experimentally infected in two quarters with 1 x 10(4) cfu Escherichia coli per quarter and six cows were infused with 500 microg endotoxin into two quarters. Six cows infected intramammarily with Esch. coli were treated intravenously with a bactericidal antibiotic 10 h after infection and subcutaneously 20 h later. Blood and milk samples were collected from all cows at regular time intervals. Milk production decreased more rapidly, but was less pronounced, after endotoxin infusion than (during Esch. coli mastitis. The milk production losses in the noninflamed quarters were negligible in endotoxin mastitis, but were substantial during Esch. coli mastitis, probably due to more pronounced systemic effects. Reticulorumen motility was inhibited only during Esch. coli mastitis. Changes in plasma haptoglobin were more pronounced during Esch. coli mastitis, although they occurred sooner during endotoxin mastitis. No changes in plasma activities of enzymes such as lactate dehydrogenase, glutamic-oxaloacetic transaminase and gamma-glutamyl transpeptidase were observed. Concentrations of tumour necrosis factor-alpha increased in both types of mastitis. Absorption of these cytokines into the circulation was highest during Esch. coli mastitis, especially in the untreated control group. We found only minor differences between the treated and untreated Esch. coli groups, but there were larger differences between the Esch. coli groups and the endotoxin group. These differences were probably due to differences in kinetics, composition and amounts of different cytokines released in the mammary gland and subsequently absorption into the circulation. Endotoxin is probably not directly responsible for the systemic changes during coliform mastitis.     

Analysis of an outbreak of Streptococcus uberis mastitis

An outbreak of Streptococcus uberis mastitis was described to gain insight into the dynamics of Strep. uberis infections at a herd level. Data were obtained from a longitudinal observational study on a commercial Dutch dairy farm with good udder health management. Quarter milk samples for bacteriological culture were routinely collected at 3-wk intervals from all lactating animals (n = 95 +/- 5). Additional samples were collected at calving, clinical mastitis, dry-off, and culling. During the 78-wk observation period, 54 Strep. uberis infections were observed. The majority of infections occurred during a 21-wk period that constituted the disease outbreak. The incidence rate was higher in quarters that had recovered from prior Strep. uberis infection than in quarters that had not experienced Strep. uberis infection before. The incidence rate of Strep. uberis infection did not differ between quarters that were infected with other pathogens compared with quarters that were not infected with other pathogens. The expected number of new Strep. uberis infections per 3-wk interval was described by means of a Poisson logistic regression model. Significant predictor variables in the model were the number of existing Strep. uberis infections in the preceding time interval (shedders), phase of the study (early phase vs. postoutbreak phase), and prior infection status of quarters with respect to Strep. uberis, but not infection status with respect to other pathogens. Results suggest that contagious transmission may have played a role in this outbreak of Strep. uberis mastitis.     

Changes in milk protein fraction as affected by subclinical mastitis

From cows that had both healthy quarters and quarters with subclinical mastitis [somatic cell count (SCC) 84,000 vs. 293,000/ml in bucket milk], foremilk, bucket milk, and stripping and residual milks were collected. Young milk was obtained 1.5 h later following a repeated oxytocin injection. Compared with milk from healthy quarters, milk from quarters with subclinical mastitis showed elevated SCC, plasminogen, and protein and had increased activity of n-Acetyl-beta-D-glucosaminidase (NAGase) and plasmin, as well as elevated portions of whey proteins and gamma-casein in the total protein. The SCC and the other mentioned parameters were also higher in the foremilk and the stripping and residual milks compared with bucket milk, independent of the udder health status; however, decreased values were found for total protein. Young milk showed an increase of SCC and n-Acetyl-beta-D-glucosaminidase activity compared with bucket milk. Because of lower levels of total plasmin and gamma-casein, we concluded that this young milk was newly synthesized milk containing some casein degradation products and that proteolysis of casein continued in the udder until the next milking. The n-Acetyl-beta-D-glucosaminidase activity was shown to be a better indicator for subclinical mastitis and correlated better with protein degradation than did SCC.     

Usability of bacteriological milk analyses for genetic improvement of udder health in Austrian Fleckvieh cows

In addition to somatic cell count records and clinical mastitis diagnoses, results of bacteriological milk analyses provide valuable information regarding udder health. The pathogen causing an udder infection is currently not considered in Austria as part of the information used for estimation of routine breeding values for mastitis resistance. Therefore the objective of this study was to estimate heritabilities for, and genetic correlations between, udder traits of bacterial infection (bacterial infection, gram-positive and gram-negative bacterial infection) and routinely recorded udder health traits [acute mastitis, chronic mastitis, culling due to udder health problems, and somatic cell score (SCS)] in Austrian Fleckvieh cows. The basis for the genetic analyses was a data set with results from bacteriological milk analyses collected from 237 dairy farms and 6,822 cows over a period of 1 yr. Traits were defined as binary, apart from SCS, for which measures were available continuously. Multivariate analyses using a linear animal model were applied for estimating genetic parameters. The heritabilities for the occurrence of bacterial udder infection traits were 0.01. Heritabilities were 0.04 for acute mastitis, 0.02 for chronic mastitis, 0.02 for culling due to udder health problems, and 0.20 for SCS. Genetic correlations between bacteriological infection and the routinely recorded udder health traits were positive and ranged from 0.62 to 0.96. The genetic correlation between gram-positive and gram-negative bacterial infection was ?0.20. The genetic correlation between acute and chronic mastitis was also close to zero. These results show that mastitis caused by different pathogens may be seen as different traits. As analyses were based on a relatively small data set and results were associated with rather high standard errors, further research with a larger data set should be carried out to confirm these results.     

Increased nuclear factor kappaB activity in milk cells of mastitis-affected cows

Bacterial mastitis is accompanied by a drastic increase in milk somatic cell count (SCC), with neutrophils being the predominant cell type found in the infected quarters. Accumulation and activation of neutrophils at the site of infection require local expression of many inflammatory genes encoding adhesion molecules, chemokines and cytokines. Most of the inflammatory genes contain binding sites for the nuclear factor kappaB (NF-kappaB) within their promoter and therefore partly depend on NF-kappaB for their expression. We thus hypothesized that an increase in NF-kappaB activity in the mammary gland could contribute to development of the neutrophilic inflammation that characterizes mastitis. In an attempt to verify this hypothesis, we first assessed milk cells from healthy and acute and chronic mastitis-affected cows for NF-kappaB activity using electrophoretic mobility shift assays. We next studied the relationships between the intensity of NF-kappaB activity in these cells and the degree of udder inflammation. Active NF-kappaB complexes were undetectable in milk cells from healthy cows, whereas high levels of NF-kappaB activity were always found in cells from cows with acute mastitis. In milk cells obtained from chronic mastitis-affected cows, NF-kappaB activity varied from low to high. Finally, the level of NF-kappaB activity measured in milk cells from chronic mastitis-affected cows was not correlated to SCC or to the proportion of neutrophils present in milk samples, but was highly correlated with the expression level of interleukin-8 and granulocyte/macrophage colony-stimulating factor, two NF-kappaB-dependent cytokines crucially involved in initiation and perpetuation of neutrophilic inflammation. These results suggest that NF-kappaB might play a role in mastitis pathogenesis.