Growth of Escherichia coli in milk from endotoxin-induced mastitic quarters and the course of subsequent experimental Escherichia coli mastitis in the cow

The objective of this study was to assess growth of Escherichia coli in milk from endotoxin-induced mastitic quarters and to relate the in vitro findings to the course of experimental E. coli mastitis. Whole and skim milks from 24 rear quarters of 12 cows were inoculated with E. coli 0:157 and incubated at 38 degrees C. Growth of E. coli 0:157 was not inhibited in milk collected from rear quarters immediately prior to endotoxin infusion. However, growth inhibition occurred in all but one whole mastitic milk samples collected from mastitic quarters 18 h after infusion of .1 mg of endotoxin. Skim milk samples from mastitic quarters were bactericidal in four cows (7 quarters), whereas growth occurred in skim mastitic milk from 17 quarters of nine cows. Rear quarters of all cows were inoculated with 10(4) cfu of E. coli 0:157 19 h after the quarters had been infused with endotoxin. Clinical parameters and milk production were monitored during 36 h and 21 d, respectively. None of the inoculated quarters developed signs of inflammation, and secreta from inoculated quarters were bacteriologically negative after 48 h. Therefore, growth-inhibitory property of skim milk from endotoxin-induced quarters was apparently not a suitable parameter to monitor differences in susceptibility to E. coli mastitis.     

Variation in the composition of selected milk fraction samples from healthy and mastitic quarters, and its significance for mastitis diagnosis

Seven variables--electrical conductivity (EC), somatic cell count (SCC), N-acetyl-beta-D-glucosaminidase (NAGase), lactose, protein, fat and pH--were compared in four quarter milk fractions (MF1: strict foremilk; MF2: first 12-15 ml foremilk; MF3: subsequent 40-45 ml milk; MF4: strippings) and in one cow composite milk sample (CC) per cow. The study used 142 quarters from 37 lactating cows of the German Black Pied breed. To rule out any possible effect due to management, animal physiology and analytical procedures, the collection and processing of milk samples from each cow was repeated for three consecutive days, and the means of 3-d values were used. All variables were affected significantly by milk fraction and udder health. Compared with foremilk, EC, lactose and protein levels in strippings decreased, while SCC, NAGase and fat increased. The pH of foremilk and strippings did not differ significantly in healthy or in mastitic quarters. The difference between MF1 and MF2 was significant for EC in mastitic quarters, and for SCC in healthy quarters only. In general, mastitis resulted in a significant increase in EC, SCC, NAGase and protein but in a decrease in lactose and fat contents of milk in one or more of the milk fractions studied. Comparison of cow composite milk samples from healthy and mastitic cows revealed the significance (P < 0.01) of udder health for EC, SCC and lactose. Of the different parameters that can distinguish between healthy and mastitic quarters or cows, EC could be used to classify 76% of quarters and 73% of cows correctly, while the lactose content permitted correct identification of 81% of quarters and 76% of cows. NAGase and pH could be used to determine the status of 73% and 61% of quarters, respectively. In general, the correlation observed in strippings was higher than in foremilk for almost all the variables studied. Surprisingly, EC, SCC, NAGase and lactose in milk from healthy quarters of mastitic cows (with at least one mastitic quarter) differed significantly (P < 0.05) from those from healthy quarters of cows with all four healthy quarters, indicating an inconsistent effect of mastitic quarters on neighbouring healthy quarters (quarter interdependence).     

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.     

Short communication: Use of an ultrafiltration device in gland cistern for continuous sampling of healthy and mastitic quarters of lactating cattle for pharmacokinetic modeling

Pharmacokinetic studies of the drugs in the milk are often limited due to infrequent sampling associated with milking. Alternatively, frequent sample collection with repeated milking may increase drug elimination. The objective of this study was to determine the feasibility of continuously sampling the udder using ultrafiltration. An ultrafiltration probe was placed into the gland cisterns through mammary parenchyma of normal and mastitic quarters of 6 mature mid-lactation Jersey cows with naturally occurring subclinical mastitis. An ultrafiltration probe was secured to the caudal or lateral aspect of the udder depending on the quarter being sampled. The timed interval samples were collected at 0, 2, 4, 6, 8, 12, 18, 24, 28, 32, 36, 48, 60, 72, 84, and 96 h after drug administration. Plasma samples were collected at the same time points. Each cow received 2.2 mg/kg of flunixin intravenously before milking at time 0. All cows were routinely milked by machine every 12 h. Flunixin concentrations in plasma, whole milk, and milk ultrafiltrates were analyzed by use of ultra-high-performance liquid chromatography with mass spectrometric detection. We found no significant effects on the appearance of the milk or the ability to milk the cows after implantation of the ultrafiltration probes. The concentration of flunixin collected from the ultrafiltration probes in the mastitic quarters tended to be greater than that of the healthy quarters. We concluded that collection of ultrafiltration samples from the mammary gland of cows provides a viable means to continuously assess drug concentrations in the milk while continuing to milk the cow normally. This study demonstrates the utility of continuous sampling of milk via ultrafiltration for future pharmacokinetic studies in cattle.     

Estimating degree of mastitis from time-series measurements in milk: a test of a model based on lactate dehydrogenase measurements

The aim of this study was to test a model for mastitis detection using a logic that allows examination of time-related changes and a progressive scale of mastitis state (i.e., not using specificity/sensitivity). The model produces a mastitis risk (MR) for individual cows on a scale from 0 (completely healthy) to 1 (full-blown mastitis). The main model input was lactate dehydrogenase (LDH; μmol/min per L) × milk yield. Test data containing 253 mastitis cases were used. Proportional samples were collected from each cow at each milking and analyzed for LDH and somatic cell count (SCC). The basis for the health definitions was veterinary treatment records. A refinement of the basic health definitions was made using systematic positive deviations in log(SCC) to indicate untreated infections. Two subsets of cows were identified: mastitic cows and cows completely free of mastitis (healthy controls). The time-profiles of these 2 groups in a 60-d window relative to day of veterinary treatment were examined. Model reliability throughout all stages of lactation and degrees of infection was examined using SCC as a continuous measure of degree of mastitis. The time-profile for the health controls was flat throughout the 60-d window with a median MR of 0.02. In contrast, the profile of the mastitic cows increased above the control cows’ baseline from about -6 d, rising to a MR value of 0.20 at d 0, and declining to the control level after treatment. There were significant differences between mastitic and healthy cows from -4 to +2 d relative to veterinary treatment. When cases were time-aligned to peak of infection, rather than veterinary treatment, there was a much sharper peak to the time-profile of mastitic cows. The median MR at peak was 0.62 and the mean was 0.80. Using these data, the MR value of 0.62 had a <1% likelihood of actually coming from a healthy control. Testing against SCC, on the whole data set, showed that only 2.1% of all MR values had an error >0.7. These estimates of model reliability are comparable with the greatest values reported in the literature and, additionally, the model was able to detect significant differences between mastitic and healthy cows 4 d before treatment. It was also found that specificity/sensitivity calculations are inappropriate for evaluating time-related changes and a progressive scale of predicted mastitis state.     

Determinants of bacterial replication rates in mastitic whey

Bacterial growth was measured by a turbidimetric microtechnique in the whey of milk samples from quarters of cows with subclinical mastitis. Samples were grouped according to bacterial isolates recovered and the effects of bacterial species and whey on bacterial growth rates were analysed. Different strains of bacteria and different whey samples gave highly significant differences in bacterial replication rates. Except for penicillin-resistant Staphylococcus aureus, bacteria grew better in whey from mastitic milk where the inflammation was caused by the same bacterial species than in other mastitic milk samples. Inflammation caused by major pathogens generally enhanced the growth in whey of any type of major pathogen. Since mastitis pathogens showed enhanced growth in whey prepared from the same milk from which they were isolated, specific antibacterial factors in the whey did not appear to restrict bacterial growth in whey. The nutritional quality of the medium seems to be the important determinant of bacterial growth.     

Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry MSE-based untargeted milk metabolomics in dairy cows with subclinical or clinical mastitis

In this study, a novel metabolomics technique based on ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry in the MS^E mode was used to investigate the milk metabolomics of healthy, subclinical, and clinical mastitis cows, which were classified based on somatic cell count and presentation of clinical symptoms. Meanwhile, univariate and multivariate statistical analyses were performed to identify the significant differences across the 3 groups. Compared with healthy milk samples, less glucose, d-glycerol-1-phosphate, 4-hydroxyphenyllactate, l-carnitine, sn-glycero-3-phosphocholine, citrate, and hippurate were detected in the clinical mastitic milk samples, whereas less d-glycerol-1-phosphate, benzoic acid, l-carnitine, and cis-aconitate were found in the subclinical mastitic milk samples. Meanwhile, the milk concentration of arginine and Leu-Leu increased in both the clinical and subclinical mastitis groups. Besides, less 4-hydroxyphenyllactate, cis-aconitate, lactose, and oxoglutarate were detected in the clinical than the subclinical mastitic milk samples, whereas the abundance of some oligopeptides (Leu-Ala, Phe-Pro-Ile, Asn-Arg-Ala-Ile, and Val-Phe-Val-Tyr) increased by over 7.95-fold. Our results suggest that significant variations exist across healthy and mastitis cows. The current metabolomics approach will help in better understanding the pathobiology of mastitis, although clinical validation will be required before field application.     

Short communication: Outbreak of Nocardia neocaledoniensis mastitis in an Italian dairy herd

Nocardia spp. are an uncommon cause of mastitis, and outbreaks have typically been reported in dairy farms with poor hygienic and management conditions. The outbreak described herein involved a dairy farm with 43 lactating cows that, after a long period with low bulk milk somatic cell counts (<180,000 cells/mL), experienced an increasing incidence of clinical mastitis with bulk milk somatic cell counts greater than 300,000 cells/mL. Fifteen mastitic quarters milk samples from 9 dairy cows were found to be infected by a member of the genus Nocardia, as identified on the basis of selected phenotypic and chemotaxonomic characteristics. The isolates were confirmed as Nocardia neocaledoniensis by 16S rDNA gene sequencing. Average quarter milk somatic cell count for infected udders was 863,057 cells/mL, significantly greater than the average value in noninfected quarters (189,710 cells/mL).     

Evaluation of the Delvo-X-Press assay for detecting antibiotic residues in milk samples from individual cows.

Performance of the Delvo-X-Press beta-lactam antibiotic assay was examined using bulk-tank milk samples and milk samples from individual cows. Bulk-tank milk samples fortified with bovine lactoferrin at a concentration of 1 mg/ml or more consistently tested positive. False-positive results were also obtained from bulk-tank milk samples fortified with bovine plasma at concentrations of 20 and 40%. The assay yielded positive results for milk with antibiotic concentrations as low as 2 ppb. Individual milk samples were collected from 144 healthy lactating cows and from 34 cows with chronic Staphylococcus aureus mastitis. Specificity estimates for samples from healthy and mastitic cows were 0.88 (95% confidence interval [CI], 0.82, 0.93) and 0.94 (95% CI, 0.86, 1.00), respectively. Individual milk samples were collected from three cows with experimentally induced mastitis for 21 consecutive days. False-positive results occurred as late as 12 days postchallenge. A moderate but significant (P < 0.01) positive linear correlation (r = 0.61) was observed between test result and somatic cell count (SCC) values in milk samples with SCCs of >10(6)/ml.     

Yield losses associated with clinical mastitis occurring in different weeks of lactation

The effects of the first lactational incidence of clinical mastitis (CM) on milk, fat, and protein production were studied in the Swedish Red and Swedish Holstein breeds. The data consisted of 38,535 weekly production records from 1,192 lactations (506 cows), sampled from 1987 to 2004 in one of the university's research herds. Daily yields were analyzed using a repeated-measures mixed model with an interaction between mastitis index and lactational stage, breed, parity, reproductive status, year-season of calving, and various indices for other disorders as independent variables. The indices were used to distinguish between cows with and without the studied diagnoses, as well as to indicate time (test day) in relation to day of diagnosis. Inclusion of the interaction made it possible to study the effects of CM occurring in different weeks of lactation. Primiparous and multiparous cows were analyzed separately, and the yields of nonmastitic cows were used as a reference for the production level in healthy cows. Lactational (305-d) yield losses were extrapolated from the daily estimates. High milk yield was predisposing to CM. Daily milk yield started to decline 2 to 4 wk before diagnosis. On the day of clinical onset, the milk yield of mastitic cows was reduced by 1 to 8 kg. After a case of CM, milk yield was suppressed throughout lactation. The magnitude of the yield losses was determined by the week of lactation at clinical onset. The greatest losses occurred when primiparous cows developed CM in wk 6, whereas multiparous cows experienced the greatest losses when diseased in wk 3. The 305-d milk, fat, and protein production in mastitic primiparous cows were reduced by 0 to 9, 0 to 8, and 0 to 7%, respectively. The corresponding reductions in mastitic multiparous cows were 0 to 11, 0 to 12, and 0 to 11%, respectively.     

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.     

Udder firmness as a possible indicator for clinical mastitis

Swelling of the mammary gland is an important sign to detect clinical mastitis (CM) in dairy cows. The overall objective of this study was to evaluate if udder firmness can be used as a cow-side indicator for mastitis and to evaluate how CM affects firmness within 14 d after diagnosis. A dynamometer was used to objectively determine udder firmness before and after milking in 45 cows with CM and 95 healthy cows. Udder firmness of both hind quarters was measured daily on 3 locations (upper, middle, lower measuring point) from the day of mastitis diagnosis until d 7 and again on d 14. Firmness of the middle measuring point was highest before and after milking in all cows. Udder firmness before milking was similar in quarters without and with CM. Subsequently, we concentrated on firmness measured on the middle point after milking. After milking, quarters with CM were firmer than healthy quarters. An increase of firmness of a quarter with mastitis did not affect firmness of the healthy neighboring quarter, nor did firmness of all healthy quarters differ. One firmness value per cow [i.e., Δ firmness (difference in udder firmness between both hind quarters)] was used for all further calculations. After fitting a generalized mixed model, CM affected Δ firmness in all cases. In multiparous cows, Δ firmness was also affected by continuous milk yield per day and DIM. Firmness thresholds for detection of CM were calculated using receiver operation characteristic curves. The threshold for detection of CM using Δ firmness was 0.282 kg (area under the curve: 0.722, sensitivity: 64.3%, specificity: 89.7%) and 0.425 kg (area under the curve: 0.817, sensitivity: 62.5%, specificity: 96.7%) in primiparous cows and multiparous cows, respectively. Linear mixed-model ANOVA were used to evaluate how CM affects udder firmness within 14 d after diagnosis. Cows with CM had a higher Δ firmness compared with cows without CM throughout the 14 d after the mastitis diagnoses. Parity had an effect on Δ firmness. Depending on systemic signs of sickness, mastitic cows were divided into cows having mild to moderate (n = 21) or severe mastitis (n = 24). Cows with severe mastitis suffered from a firmer udder on all measuring days. Bacteriological cure was defined based on 2 milk samples taken at 7 and 14 d after enrollment. An effect of parity and bacteriological cure on Δ firmness of cows with CM did not exist within the 14 d. Cows not clinically cured showed an increased Δ firmness of 0.560 kg compared with cured cows. In conclusion, udder firmness can be a useful indicator for CM. Further research is warranted to evaluate if udder firmness can be used as a predictor for the prognosis of a CM or the cure of inflammation.     

The hidden cost of disease: I. Impact of the first incidence of mastitis on production and economic indicators of primiparous dairy cows

Mastitis is a highly prevalent disease, which negatively affects cow performance, profitability, welfare, and longevity. The objectives of this study were (1) to quantify the impact of the first instance of mastitis, at different stages of lactation, on production and economic performance, and (2) to further quantify the impact of the first instance of mastitis when only cows that remain in the herd for at least 100 d in milk (DIM) and those that remain for 305 DIM are included in the analysis. A retrospective longitudinal study was conducted using data from existing animal health record files and Dairy Herd Improvement records. After editing based on selected inclusion criteria and completeness of health records, data consisted of records from first-lactation Holstein cows, from 120 herds, that calved for the first time between 2003 and 2014, inclusive. Mastitic cows were assigned to 1 of 4 groups based on when in the lactation the first event of mastitis occurred: transition (1–21 DIM), early lactation (22–100 DIM), mid lactation (101–200 DIM), or late lactation (201+ DIM). Mid-lactation and late-lactation mastitic cows were also stratified by cumulative milk yield before the mastitis event. Healthy cows (i.e., no recorded mastitis event) were randomly assigned for each lactation stage, with mid-lactation healthy and late-lactation healthy cows similarly stratified. Production performance (cumulative milk, fat, and protein yield) and economic performance [milk value, margin over feed cost (MOFC), and gross profit] were analyzed using a mixed model with herd as a random effect. Significant losses in cumulative milk yield (?382 to ?989 kg) and correspondingly lower fat and protein yields were found in mastitic cows, with transition and late-lactation mastitic cows having the highest losses. Drops in production translated to significant reductions in cumulative milk value (?Can$287 to ?Can$591; ?US$228 to ?US$470), MOFC (?Can$243 to ?Can$540; ?US$193 to ?US$429), and gross profit (?Can$649 to ?Can$908; ?US$516 to ?US$722) for mastitic cows at all stages. Differences between mastitic and healthy cows in the early lactation and transition stages remained for all variables in the 100-DIM analysis, but, aside from gross profit, were nonsignificant in the 305-DIM analysis. Gross profit accounted for all costs associated with mastitis and thus continued to be lower for mastitic cows at all stages, even in the 305-DIM analysis in which culled cows were omitted (?Can$485 to ?Can$979; ?US$386 to ?US$779). The research reflects the performance implications of mastitis, providing more information upon which the producer can make informed culling decisions and maximize both herd profitability and cow longevity.     

Haptoglobin and serum amyloid A in milk and serum during acute and chronic experimentally induced Staphylococcus aureus mastitis

Local and systemic changes in the acute phase proteins, haptoglobin and serum amyloid A (SAA), were studied in six dairy cows during the acute and chronic phases of experimentally induced Staphylococcus aureus mastitis. Haptoglobin and SAA were measured in serum, and in milk from infected and healthy control udder quarters within each cow. Concentrations of haptoglobin and SAA increased rapidly in both serum and milk during the acute phase of mastitis and followed a similar pattern. Significantly raised milk concentrations of SAA were also found during chronic subclinical mastitis. Serum concentrations of SAA also tended to be higher during the chronic phase than pre-infection. Increases in milk haptoglobin and SAA were specific for the infected udder quarters. In conclusion, measurement of SAA in milk samples could be a useful tool in diagnosing mastitis.     

Somatic cell count status across the dry period as a risk factor for the development of clinical mastitis in the subsequent lactation

The objective of this study was to determine the risk of clinical mastitis in the first 120 d in lactation based on previous somatic cell count (SCC) history in a herd with a very low prevalence of contagious pathogens. A total of 218 cows from a university herd were enrolled at dry-off. Duplicate quarter milk samples were collected from all quarters at dry-off, postcalving (2 to 9 d in milk), and before treatment of all first cases of clinical mastitis that occurred during the first 120 d of the subsequent lactation. Quarter SCC statuses across the dry period were defined based on comparison of quarter SCC between the date of dry-off and the postcalving sampling periods. The relationship between the probability of developing clinical mastitis in the first 120 d of lactation and SCC status across the dry period and other explanatory variables was assessed using logistic regression. In the first 120 d postcalving, 68 first cases of clinical mastitis occurred in 47 cows. Of quarters that experienced a microbiologically positive clinical case, the same microorganism was never isolated from milk samples obtained at dry-off or consistently isolated from milk samples collected at all sampling periods. Coagulase negative staphylococci were the most prevalent pathogens isolated from subclinical intramammary infection, whereas gram-negative pathogens were the most common pathogen associated with clinical cases. Quarters that had at least 1 case of mastitis during the previous lactation were 4.2 times more likely to have a first case of clinical mastitis in the current lactation than quarters that did not have clinical mastitis in the previous lactation [odds ratio (OR) = 4.2 (1.8, 10.0)]. Quarters of cows of greater than fourth parity were 4.2 times more likely to have a first case of clinical mastitis than quarters of cows of second parity [OR = 4.2 (1.4, 10.0)]. Quarters with SCC ≥200,000 cells/mL at dry-off and postcalving were 2.7 times more likely to experience a first case of mastitis than quarters with SCC <200,000 cells/mL at both periods [OR = 2.7 (0.97, 7.67)].     

Evaluation of automatic mastitis detection equipment

An electronic sensor was evaluated as an instrument for early detection of mastitis. This method involved measuring the conductivity of milk continuously throughout the milking process and then establishing a conductivity ratio. The lowest conductivity measurement of the four quarters was a basis for assessing the degree of mastitis in the other quarters. This assumed that at least one of the quarters was normal at examination and the lowest reading was normal conductivity. The conductivity ratio was evaluated by comparison with the leukocyte concentration and combined leukocyte concentrations and cultural examiniations of milk samples from 1028 quarters. In healthy cows conductivities of milk from each of the quarters were similar. If, however, one or more quarters were infected, this milk showed higher conductivity compared to the noninfected quarter of the same cow. The conductivity ratio correctly identified 69% of the established cases of mastitis. For the Wisconsin Mastitis Test, 93.2% of the normal quarters were detected correctly by the conductivity ratio. Leukocyte counts were frequently high when there was no other evidence of mastitis. We believe the conductivity ratio is effective in detecting mastitis at an early stage of infection caused by most of the pathogenic microorganisms.     

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.     

Suppression of milk production during endotoxin-induced mastitis.

Healthy, midlactation cows were given intramammary infusions of 10 micrograms of endotoxin in two homolateral quarters. Productive, inflammatory, and systemic responses were studied to investigate the pathophysiological effects of mastitis on lactational performance. Endotoxin suppressed milk yield in all quarters of treated cows. A more severe and prolonged suppression occurred in infused quarters compared with uninfused quarters. The fat percentage of milk from all quarters was increased with a greater increase occurring in infused quarters. The protein composition of milk was elevated, and the lactose concentration was depressed in infused quarters. Mammary inflammation--as measured by milk SCC, NAGase, serum albumin, and lactoferrin--was limited to infused quarters. Changes in milk NAGase closely paralleled changes in milk SCC. Daily feed intake was unaffected, and serum glucose levels did not decline following infusion. The lactose concentration of urine increased rapidly after infusion. Reduction in milk yield in all quarters, but varying changes in milk composition in infused versus uninfused quarters suggest that mastitic hypogalactia is mediated by multiple pathophysiological events and is not solely due to inflammatory damage to the mammary epithelium. Part of the reduced lactational performance may result from escape of milk components from the udder into the circulation.