Lactoperoxidase potential in diagnosing subclinical mastitis in cows via image processing.

This report describes how image processing harnessed to multivariate analysis techniques can be used as a bio-analytical tool for mastitis screening in cows using milk samples collected from 48 animals (32 from Jersey, 7 from Gir, and 9 from Guzerat cow breeds), totalizing a dataset of 144 sequential images was collected and analyzed. In this context, this methodology was developed based on the lactoperoxidase activity to assess mastitis using recorded images of a cuvette during a simple experiment and subsequent image treatments with an R statistics platform. The color of the sample changed from white to brown upon its exposure to reagents, which is a consequence of lactoperoxidase enzymatic reaction. Data analysis was performed to extract the channels from the RGB (Red-Green-Blue) color system, where the resulting dataset was evaluated with Principal Component Analysis (PCA), Multiple Linear Regression (MLR), and Second-Order Regression (SO). Interesting results in terms of enzymatic activity correlation (R2 = 0.96 and R2 = 0.98 by MLR and SO, respectively) and of somatic cell count (R2 = 0.97 and R2 = 0.99 by MLR and SO, respectively), important mastitis indicators, were obtained using this simple method. Additionally, potential advantages can be accessed such as quality control of the dairy chain, easier bovine mastitis prognosis, lower cost, analytical frequency, and could serve as an evaluative parameter to verify the health of the mammary gland.

Involvement of matrix metalloproteinases and their inhibitors in Staphylococcus aureus chronically infected bovine mammary glands during active involution.

The aim of this study was to characterize the protein expression of matrix metalloproteinase-2 (MMP-2) and -- 9 and their inhibitors (TIMP-1 and -2) in mammary tissue of dairy cows with naturally occurring chronic S. aureus intramammary infections (IMI) during active involution. Moreover, the gelatinolytic activity of MMP-2 and -9 in mammary secretions was evaluated. Cows in late lactation that were either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. Protein expression of MMP-2 and -9 in mammary tissues was significantly higher in S. aureus-infected than uninfected quarters at day 14 and 21 of involution. Protein expression of TIMP-1 and -2 was significantly higher in S. aureus-infected than uninfected quarters at day 7, 14 and 21 of involution. The MMP-2/TIMP-1, MMP-2/TIMP-2, MMP-9/TIMP-1 and MMP-9/TIMP-2 ratios were significantly higher in S. aureus-infected compared with uninfected quarters at day 14 of involution. The MMP-2 activity was significantly higher in mammary secretions from S. aureus-infected compared with uninfected quarters at day 1, 2, 7 and 14 of involution. The MMP-9 activity was significantly higher in mammary secretions from infected quarters compared with uninfected quarters at day 7, 14 and 21 of involution. The increased expression of MMP-2 and -9 in mammary tissue as well as the high levels of activity observed in mammary secretion from infected quarters compared with uninfected quarters during active involution, strongly suggests that these gelatinases could contribute to degradation of mammary tissue components during chronic S. aureus IMI. The MMPs/TIMPs imbalance could lead to greater proteolysis and potentially more damage to mammary tissue in S. aureus-infected quarters.

High expression of aldehyde dehydrogenase 1 and tissue necrosis factor alpha may relate to chronic infection of buffalo mammary gland.

Aldehyde dehydrogenase 1 (ALDH1) and hepatocyte nuclear factor 4A (HNF4A) are the putative mammary stem cell markers. Tissue necrosis factor alpha (TNFA) is involved in inflammation-associated carcinogenesis and cell proliferation. In this study, the gene expression profile of ALDH1, HNF4A and TNFA of buffalo mammary tissue using real-time quantitative PCR (RT-qPCR). Analysis of RT-qPCR data revealed that the relative expression (log2 fold change) of ALDH1 and TNFA during mastitis (vs. lactation) was increased (P < .05) by 2.98 and 4.71, respectively. The relative expression (log2 fold change; -7.39) of stem cell marker, HNF4A was decreased (P < .05) during mastitis. Histological analysis of mammary tissue during mastitis showed thickening of stroma and occasionally hyperplasia, predominantly in prepubertal and non-lactating animals. Although, the level of expression of these genes may vary, depending upon the physiological stage of the animals, however expression of ALDH1 and TNFA was high during mastitis. A systematic study on large samples of buffalo mammary tissue with appropriate comparisons needs to be evaluated with these markers for prognosis of buffalo mammary health.

Epidemiology of ß-Lactamase-Producing Staphylococci and Gram Negative Bacteria as Cause of Clinical Bovine Mastitis in Tunisia.

The aim of this study was to determine the species distribution of Staphylococcus, Gram negative bacteria (GNB) and the occurrence of Methicillin Resistant Staphylococci (MRS) and Extended-Spectrum ß-lactamase- (ESBL-) producing GNB. Bacterial culture of 300 clinical mastitis milk samples from 30 different farms across different regions of Tunisia during four seasons was realized. The obtained results showed the presence of high frequency of the tested samples with a positive growth for bacteria (64%). In addition a high recovery rate of Staphylococci and/or GNB in these clinical mastitis milk samples (87%) was detected. In addition, a high percentage of GNB (68.2%) compared to Staphylococcus species (32%) was noted. Moreover, a significant variation of the number of these bacteria according to the farm location, the seasons, and cows age was detected. The highest percentage was observed in the North of Tunisia during the winter and the spring seasons in adult cows with a dominance of GNB growth. Coagulase negative Staphylococci (CNS) (n=11) and GNB (n=16) species were identified. Escherichia coli (E. coli) was the most frequently found bacterium followed by Klebsiella pneumoniae. The dominant Staphylococcus isolates was S. xylosus followed by S. aureus the major pathogen isolated. Methicillin resistance was confirmed by the presence of the mecA gene in 3 S. aureus and 14 CNS isolates; all of these isolates were lacking the mecC gene. Various species of GNB, resistant to cefotaxime, were detected (n=15). ESBLs were detected on selective medium in 10 E. coli and 4 K. pneumoniae. All ESBL producers strains carry the blaCTX-M. The presence of different resistant mastitis pathogens in dairy farms may complicate therapeutic options and contaminated animals could become zoonotic agent reservoir for human.

HDAC1/2-mediated regulation of JNK and ERK phosphorylation in bovine mammary epithelial cells in response to TNF-α.

Bovine mammary epithelial cells (MAC-Ts) are a common cell line for the study of mammary epithelial inflammation; these cells are used to mechanistically elucidate molecular underpinnings that contribute to bovine mastitis. Bovine mastitis is the most prevalent form of disease in dairy cattle that culminates in annual losses of two billion dollars for the US dairy industry. Thus, there is an urgent need for improved therapeutic strategies. Histone deacetylase (HDAC) inhibitors are efficacious in rodent models of inflammation, yet their role in bovine mammary cells remain unclear. HDACs have traditionally been studied in the regulation of nucleosomal DNA, in which deacetylation of histones impact chromatin accessibility and gene expression. Using MAC-T cells stimulated with tumor necrosis factor α (TNF-α) as a model for mammary cell inflammation, we report that inhibition of HDACs1 and 2 (HDAC1/2) attenuated TNF-α-mediated inflammatory gene expression. Of note, we report that HDAC1/2-mediated inflammatory gene expression was partly regulated by c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation. Here, we report that HDAC1/2 inhibition attenuated JNK and ERK activation and thus inflammatory gene expression. These data suggest that HDACs1 and 2 regulate inflammatory gene expression via canonical (i.e., gene expression) and noncanonical (e.g., signaling dependent) mechanisms. Whereas, further studies using primary cell lines and animal models are needed. Our combined data suggest that HDAC1/2-specific inhibitors may prove efficacious for the treatment of bovine mastitis.

Evaluation of 3 esterase tests for the diagnosis of subclinical mastitis at dry-off and freshening in dairy cattle.

Subclinical mastitis (SCM) and intramammary infection (IMI) increase esterase activity in the glandular secretions of dairy cattle. Our objective was to evaluate the clinical performance of 3 commercially available esterase tests for diagnosing SCM and IMI. Foremilk samples were collected from 380 quarters (96 cows) at dry-off and from 329 quarters (83 cows) within 4 to 7 d after calving. Quarter somatic cell count (SCC) was measured using the reference method (DeLaval cell counter; De Laval International AB, Tumba, Sweden) with SCM defined as SCC >200,000 cells/mL. Bacterial culture of foremilk samples was used to diagnose IMI based on the growth of ≥100 cfu/mL. The SCC was estimated using 3 PortaSCC tests (PortaCheck, Moorestown, NJ) from the measured esterase activity and the California Mastitis Test (CMT). Clinical performance was evaluated using logistic regression to determine the area under the receiver operating characteristic curve (AUC) and identify test sensitivity (Se) and specificity (Sp) at the optimal cut-point for diagnosing SCM and IMI. Test agreement was also evaluated using the kappa coefficient (κ) and weighted κ. The PortaSCC color test was the best-performing PortaSCC test for diagnosing SCM at dry-off (AUC = 0.90, Se = 0.91, Sp = 0.81, κ = 0.71) and at freshening (AUC = 0.86, Se = 0.74, Sp = 0.95, κ = 0.72), at an optimal cut-point of ≥250,000 cells/mL but required 45 min to produce a result. For comparison, the CMT required 2 min to produce a result and a CMT score of trace or higher was superior to the PortaSCC color test for diagnosing SCM at dry-off (AUC = 0.95, Se = 0.95, Sp = 0.86, κ = 0.81) and freshening (AUC = 0.88, Se = 0.79, Sp = 0.95, κ = 0.76). The PortaSCC quick test was the best-performing PortaSCC test for diagnosing IMI at dry-off (AUC = 0.81, Se = 0.81, Sp = 0.78 κ = 0.40) and required 5 min to produce a result, whereas the PortaSCC color test was the best performing PortaSCC test for diagnosing IMI at freshening (AUC = 0.80, Se = 0.75, Sp = 0.79 κ = 0.38). For comparison, the CMT was inferior to the PortaSCC quick test for diagnosing IMI at dry-off (AUC = 0.73, Se = 0.76, Sp = 0.60, κ = 0.20) but was equivalent to the PortaSCC color test at freshening (AUC = 0.79, Se = 0.58, Sp = 0.93, κ = 0.50). The PortaSCC color and quick tests and CMT were considered good tests for diagnosing SCM and IMI because clinically useful tests typically have an AUC >0.80 and κ >0.6. Based on the test sensitivity, cost, and analysis time, there does not appear to be a persuasive reason to select the PortaSCC tests over the traditional CMT for diagnosing SCM and IMI.

Tryptophan, kynurenine, kynurenic acid concentrations and indoleamine 2,3-dioxygenase activity in serum and milk of dairy cows with subclinical mastitis caused by coagulase-negative staphylococci.

The aim of the study was to investigate serum and milk concentrations of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and indoleamine 2,3-dioxygenase (IDO) activity in cows suffering from subclinical mastitis caused by coagulase-negative staphylococci (MSCNS). TRP and kynurenines were determined by high-performance liquid chromatography (HPLC), and IDO activity was calculated as the KYN/TRP ratio. The blood and milk samples were collected from 40 midlactation Holstein-Fresian cows from two herds in the Lublin region in Poland. In the milk samples from 20 cows with subclinical mastitis, coagulase-negative staphylococci were isolated and in the milk obtained from healthy cows growth of microorganisms was not detected. TRP, KYN and KYNA concentrations were significantly lower in milk of cows with MSCNS compared to control animals (4.47 vs. 7.24 µM, 0.14 vs. 0.21 µM, 1.58 vs. 2.18 nM, respectively). There was no statistically significant difference in TRP and KYNA concentrations in serum between the studied animal groups (32.97 vs. 39.29 µM, 31.3 vs. 26.5 nM, respectively). In turn, the level of KYN was lower in the serum (0.81 vs. 1.13 µM) of cows with mastitis compared to healthy ones. No statistically significant differences in IDO activity, both in serum and in milk (25.24 and 27.55, 28.56 and 27.17, respectively) was revealed between the studied groups. These findings may have potential implications for diagnosis of mastitis in cows because reduction of these parameters in milk might be a marker predicting the occurrence of the disease.

Comparative Analysis of V-Akt Murine Thymoma Viral Oncogene Homolog 3 (AKT3) Gene between Cow and Buffalo Reveals Substantial Differences for Mastitis.

AKT3 gene is a constituent of the serine/threonine protein kinase family and plays a crucial role in synthesis of milk fats and cholesterol by regulating activity of the sterol regulatory element binding protein (SREBP). AKT3 is highly conserved in mammals and its expression levels during the lactation periods of cattle are markedly increased. AKT3 is highly expressed in the intestine followed by mammary gland and it is also expressed in immune cells. It is involved in the TLR pathways as effectively as proinflammatory cytokines. The aims of this study were to investigate the sequences differences between buffalo and cow. Our results showed that there were substantial differences between buffalo and cow in some exons and noteworthy differences of the gene size in different regions. We also identified the important consensus sequence motifs, variation in 2000 upstream of ATG, substantial difference in the "3'UTR" region, and miRNA association in the buffalo sequences compared with the cow. In addition, genetic analyses, such as gene structure, phylogenetic tree, position of different motifs, and functional domains, were performed to establish their correlation with other species. This may indicate that a buffalo breed has potential resistance to disease, environment changes, and airborne microorganisms and some good production and reproductive traits.

Association of MAP4K4 gene single nucleotide polymorphism with mastitis and milk traits in Chinese Holstein cattle.

The objective of the studies presented in this Research Communication was to investigate the association of single nucleotide polymorphisms present in the MAP4K4 gene with different milk traits in dairy cows. Based on previous QTL fine mapping results on bovine chromosome 11, the MAP4K4 gene was selected as a candidate gene to evaluate its effect on somatic cell count and milk traits in ChineseHolstein cows. Milk production traits including milk yield, fat percentage, and protein percentage of each cow were collected using 305 d lactation records. Association between MAP4K4 genotype and different traits and Somatic Cell Score (SCS) was performed using General Linear Regression Model of R. Two SNPs at exon 18 (c.2061T > G and c.2196T > C) with genotype TT in both SNPs were found significantly higher for somatic SCS. We found the significant effect of exon 18 (c.2061T > G) on protein percentage, milk yield and SCS. We identified SNPs at different location of MAP4K4 gene of the cattle and several of them were significantly associated with the somatic cell score and other different milk traits. Thus, MAP4K4 gene could be a useful candidate gene for selection of dairy cattle against mastitis and the identified polymorphisms might potentially be strong genetic markers.

N-acetyl -ß-D-glucosaminidase activity in cow milk as an indicator of mastitis.

Activity of lysosomal N-acetyl-ß-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1-1·04 pmoles 4-MU/min/µl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

Low expression of the antibacterial factor L-amino acid oxidase in bovine mammary gland.

In the mouse, L-amino acid oxidase (LAO) produces hydrogen peroxide by utilizing free amino acids and is a proven antibacterial factor in mammary glands. Mastitis, a bacterial infection of the mammary gland, is the most frequent disease in dairy cattle. Here, we investigate whether LAO is expressed in the mammary gland of dairy cattle and is antibacterial. In dairy cattle, the expression level of LAO mRNA in the mammary gland was considerably lower than that in mice, and LAO activity was not observed in cattle milk that produced hydrogen peroxide. The expression of LAO mRNA was also low in Japanese Black cattle, the same as in Holstein cattle. A higher LAO mRNA expression was observed in the mastitis glands than in the lactating glands. Furthermore, spleen and lymph nodes expressed high levels of LAO mRNA in dairy cattle. We conclude that mammary glands in dairy cattle have lower ability to express the LAO gene compared to that in mice, which may result in a high incidence of mastitis.

Polymorphisms of the ATP1A1 gene associated with mastitis in dairy cattle.

Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.

Reconstruction of metabolic network in the bovine mammary gland tissue.

Understanding bovine metabolism and its relationship with milk products is important in cow breeding. In the present work, the metabolic network in the mammary gland tissue of cattle was reconstructed with the available bovine genome information using several public datasets from NCBI, Uniprot, and KEGG. The network consisted of 1,743 metabolites named by KEGG compound numbers as nodes and 657 enzymes that catalyzed the corresponding reactions as edges. The characteristics of the network were analyzed. The top 20 hub metabolites were determined, and the mean path length was identified to be 6.52. Moreover, 11 key enzymes with significant changes in expression under the condition of mastitis were identified and analyzed by integrating the microarray expression data of normal and clinical mastitis. Aside from the GATM gene, 10 downregulated enzymes were detected in bovine with mastitis. In addition, many of the identified enzymes were involved in amino acid metabolisms or had a direct connection to amino acid metabolisms. These results indicate that mastitis could affect the expression of enzymes, which is vital in some amino acid metabolisms, resulting in the reduction of milk proteins. The present work provides information that may improve the understanding on bovine milk production and mastitis.

Optimizing the fluorometric ß-glucuronidase assay in ruminant milk for a more precise determination of mastitis.

Activity of the enzyme ß-glucuronidase (EC 3.2.1.31) is found in milk from ruminants with mastitis. However, the use of this enzymic activity as an indicator of mastitis has gained little attention possibly because of its low activity when compared with other mastitis indicators. The determination may therefore be less precise and the analytical procedure very time consuming and labour intensive. The present study optimized the fluorometric determination of the ß-glucuronidase activity with respect to substrate concentration, pH, incubation time etc., validated the assay, and developed it into large scale analyses. The assay performance is satisfactory regarding precision, linearity etc., and it appears comparable to analogous fluorometric assays for mastitis indicators in milk. From a local dairy herd, 825 milk samples were analysed for potential mastitis indicators, i.e. ß-glucuronidase, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and N-acetyl-ß-d-glucosaminidase (NAGase) activity, and for somatic cell counts (SCC) and the variables were compared. Activity of ß-glucuronidase was moderately but significantly correlated to SCC (r=0·21; n=768) as well as the other mentioned variables (r=0·25-0·43; n=825). Simple indices based on ß-glucuronidase and LDH or NAGase activity were tested as indicators of mastitis (SCC), but were not found to improve the diagnostic value. Future studies may further verify whether ß-glucuronidase can compete with well-established indicators of mastitis in cows such as LDH or NAGase as well as determine whether ß-glucuronidase activity, in combination with other indicators of mastitis, has an advantage. Nineteen milk samples from subclinical and latent cases of mastitis (individual quarters) were identified for specific pathogens (PCR method) and measured for ß-glucuronidase activity. The activity was tested at four different pH levels (5·5, 6·0, 6·5 and 7·0) in order to investigate the possibility of discrimination between pathogens. However, all milk samples (strains of pathogens) had the same pH optimum for ß-glucuronidase activity; this may indicate that enzymic activity from mammary tissue and leucocytes dominates over enzyme activity from bacterial cells.

Quantifying degree of mastitis from common trends in a panel of indicators for mastitis in dairy cows.

This paper has 2 objectives. First, it argues that it is beneficial to regard degree of infection with respect to mastitis as a latent quantity varying continuously from 0 (truly healthy) to 1 (full-blown clinical mastitis). This quantity is denoted as degree of infection (DOI). The DOI is based on extracting common characteristics from a panel of indicators measured repeatedly over time. The indicators used in this paper are electrical conductivity (EC), somatic cell count (SCC), and the immune response related enzyme lactate dehydrogenase (LDH). Second, this paper presents a statistical model for such data and a corresponding method for estimating the DOI from a panel of indicators. An empirical proof of concept is provided. Using DOI, there was a significant difference between the DOI of mastitic and healthy control cows beginning 5 d before the mastitic cows were treated for mastitis.

Short communication: can the mammopathogenic Escherichia coli P4 strain have a direct role on the caseinolysis of milk observed during bovine mastitis?

During bacterial bovine mastitis, the quality of milk is altered because of caseinolysis. Endogenous potential actors in milk responsible for this caseinolysis have been well studied, unlike the exogenous bacterial ones. The aim of this study was to evaluate the direct role in caseinolysis of a mammopathogenic strain, Escherichia coli P4. Secretion of at least 4 extracellular bacterial caseinolytic enzymes was highlighted by zymography, in 3 different growth media, and at each bacterial growth state, suggesting that their expression was constitutive. Different experimental conditions to evaluate caseinolytic potential did not show any significant caseinolytic activity of E. coli P4 and of the 4 extracellular proteases detected, suggesting that the high caseinolysis observed during E. coli bovine mastitis does result from endogenous milk actors.

Proteomic analysis of mammary tissues from healthy cows and clinical mastitic cows for identification of disease-related proteins.

To investigate the disease-related proteins and understand molecular mechanism of mastitis at the protein level, this project presents the protein changes in the mammary gland between healthy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-DE), after stained with colloidal Coomassie Bright Blue, six spots of differentially expressed protein were detected by PDQuest software and subjected to ion trap mass spectrometer equipped with a HPLC system, and five proteins were identified. Hemoglobin beta, kappa-casein and tryptophanyl-tRNA-synthetase (TrpRS) in healthy dairy cows, while hemoglobin beta, cytochrome C oxidase and annexin V in clinical mastitic cows were identified, they were involved in binding, transport and catalytic activity. The results may provide valuable information for the investigating of the host mammary immune system response to defense against pathogens at the protein level and potential protein targets for treatment.

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; mumol/min per L) x 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.

Bovine blood neutrophil acyloxyacyl hydrolase (AOAH) activity during endotoxin and coliform mastitis.

The dynamics of blood neutrophil acyloxyacyl hydrolase (AOAH) activity, the appearance of endotoxin (lipopolysaccharide, LPS) in blood and the role of blood neutrophil AOAH in the severity of Escherichia coli and endotoxin mastitis were investigated in early postpartum dairy cows experimentally challenged with either endotoxin (n = 6) or E. coli (n = 6). The AOAH activity of blood neutrophils started to decrease significantly at post challenge hours (PCH) 6-24 and 12-24 in the endotoxin and E. coli-challenged groups, respectively; it returned to pre-challenged values at PCH 48 in both endotoxin- and E. coli-challenged groups. The cows were classified as moderate and severe responders according to milk production loss in the non-challenged quarters at PCH 48. There were no severe responders in the endotoxin-challenged group. In the E. coli-challenged group, only 1 severe responder was identified. The pre-challenge neutrophil AOAH activity of the severe responder was approximately 30% lower than that of moderate responders. No LPS was detected in the plasma of endotoxin-challenged cows; neither was it found in the plasma of moderate responders in the E. coli-challenged group at any PCH. However, at PCH 6, a remarkable amount of LPS was detected in the plasma of the severe responder from the E. coli-challenged group. Furthermore, neutrophil AOAH activity was increased by approximately 70% in the severe responder at PCH 6, but it increased by only approximately 15% in moderate responders. This was followed by a decreased neutrophil AOAH activity at PCH 12-24 and 24-72 in moderate and severe responders, respectively; the decreased AOAH activity at those PCH was more pronounced in the severe responder. The pronounced decreased neutrophil AOAH activity during mastitis often coincided with extreme leukopenia, neutropenia and a maximal number of immature neutrophils in the blood. Our results demonstrate that a decrease in neutrophil AOAH activity results in the appearance of LPS in the blood, and low blood neutrophil deacylation activity could be considered as a risk factor for severe clinical coliform mastitis.

Assessment of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activities in cow's milk as an indicator of subclinical mastitis.

This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those which were classified as positive by California mastitis test (CMT) were deemed to have subclinical mastitis. All the milk samples were skimmed by centrifugation at 10 000g at 0 degrees C and were used for enzyme activities estimations. The mean activities of LDH and ALP were higher in the milk from udders with SCM than in the milk from healthy udders (p < 0.05). There were no significant differences in AST values. The maximum agreement rates between the CMT results and LDH and ALP values were seen at thresholds of > 180 IU/L and > 40 IU/L respectively (kappa values 0.65 and 0.79, respectively). However, the sensitivity of the tests for identifying SCM at these thresholds was higher for ALP (96.4%) than for LDH (68.5%). In this study, LDH and ALP tests were standardized for cow's milk and results showed that only the ALP test was reliable in the early diagnosis of subclinical mastitis.