Milk Amyloid A Research Articles

Animal and Farm Factors Affecting the Fatty Acid Profile and Amyloid A Concentration of Milk on Quebec Dairy Farms

Relationships between farm and animal factors and the fatty acid (FA) profile and milk amyloid A (MAA) content of milk were determined in 336 Holstein dairy cows on 24 Quebec farms using multiple regression. Cows with a somatic cell count (SCC) above 200,000, and farms feeding palm oil were excluded. Independent factors of the regression models included days in milk (DIM), parity, yield, fat and protein contents of milk, SCC, and the dietary contents of neutral detergent fibre (NDF) and crude fat (CFAT). Non- significant variables with P-values >0.25 were stepwise removed. Models with high fits were those of total short chain FA, medium chain FA, odd- and branched-chain FA (OBCFA), and saturated long chain FA with R2 of 0.33, 0.36, 0.34, and 0.41, respectively. The fat and protein contents and yield of milk did not affect the milk FA profile. Higher NDF increased the milk fat proportions of short chain FA and OBCFA and decreased those of monounsaturated (MUFA) and polyunsaturated FA (PUFA). Higher CFAT reduced this proportion of short chain FA but increased those of MUFA and PUFA. Increasing DIM reduced this proportion of short chain FA and increased that of medium chain FA. Higher SCC increased MAA.

Biomarker and proteome analysis of milk from dairy cows with clinical mastitis: Determining the effect of different bacterial pathogens on the response to infection

Antimicrobial usage (AMU) could be reduced by differentiating the causative bacteria in cases of clinical mastitis (CM) as either Gram-positive or Gram-negative bacteria or identifying whether the case is culture-negative (no growth, NG) mastitis. Immunoassays for biomarker analysis and a Tandem Mass Tag (TMT) proteomic investigation were employed to identify differences between samples of milk from cows with CM caused by different bacteria. A total of 94 milk samples were collected from cows diagnosed with CM across seven farms in Scotland, categorized by severity as mild (score 1), moderate (score 2), or severe (score 3). Bovine haptoglobin (Hp), milk amyloid A (MAA), C-reactive protein (CRP), lactoferrin (LF), α-lactalbumin (LA) and cathelicidin (CATHL) were significantly higher in milk from cows with CM, regardless of culture results, than in milk from healthy cows (all P-values <0.001). Milk cathelicidin (CATHL) was evaluated using a novel ELISA technique that utilises an antibody to a peptide sequence of SSEANLYRLLELD (aa49–61) common to CATHL 1–7 isoforms. A classification tree was fitted on the six biomarkers to predict Gram-positive bacteria within mastitis severity scores 1 or 2, revealing that compared to the rest of the samples, Gram-positive samples were associated with CRP < 9.5 μg/ml and LF ≥ 325 μg/ml and MAA < 16 μg/ml. Sensitivity of the tree model was 64%, the specificity was 91%, and the overall misclassification rate was 18%. The area under the ROC curve for this tree model was 0.836 (95% bootstrap confidence interval: 0.742; 0.917). TMT proteomic analysis revealed little difference between the groups in protein abundance when the three groups (Gram-positive, Gram-negative and no growth) were compared, however when each group was compared against the entirety of the remaining samples, 28 differentially abundant protein were identified including β-lactoglobulin and ribonuclease. Whilst further research is required to draw together and refine a suitable biomarker panel and diagnostic algorithm for differentiating Gram- positive/negative and NG CM, these results have highlighted a potential panel and diagnostic decision tree. Host-derived milk biomarkers offer significant potential to refine and reduce AMU and circumvent the many challenges associated with microbiological culture, both within the lab and on the farm, while providing the added benefit of reducing turnaround time from 14 to 16 h of microbiological culture to just 15 min with a lateral flow device (LFD).

Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin β 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

228 President Oral Presentation Pick: Milk biomarkers for determining the incidence of sub-acute ruminal acidosis

Abstract In order to test the validity of milk amyloid A (MAA) as a biomarker for inflammation and subacute ruminal acidosis (SARA), 320 milk samples from 24 commercial dairy farms in Quebec were tested for milk amyloid A using a commercial kit. These farms were divided into low risk of SARA farms and high risk of SARA farms according to the proportions of short chain and polyunsaturated fatty acids content in the bulk tank of the farms. It was assumed that farms at risk of SARA had a lower proportion of short chain and a higher proportion of polyunsaturated fatty acids compared to farms that were not at risk of SARA. Farms were also blocked in groups of two farms by geographical location and management. Each block included an at-risk and a not-at-risk farm. On each farm, 7 early- to mid-lactation and 7 mid- to late-lactation cows were randomly selected for MAA analysis. Cows with a somatic cell count (SCC) of over 200,000 in pooled milk samples were not included. Data were analyzed using SAS Proc. Mixed with Stage of lactation and Risk of SARA as fixed factors, and Block as a random factor. The model also included somatic cell counts (SCC) and parity as a covariates. The concentrations of MAA ranged from non-detectable, i.e. below 0.1 ug/ml, to 3267.9 ug/ml with an average of 336.28 ug/ml.The effects of Block, Stage of lactation, and Risk of SARA on MAA were not significant. However, SCC and parity were significantly (P &amp;lt; 0.01) correlated (P &amp;lt; .001) with MAA with correlation coefficients of 0.34 and 0.26, respectively. The results show that measurement of MAA may be a suitable biomarker for subclinical inflammations such as subclinical mastitis, but not for the Risk of SARA.

The Relation between Biochemical Parameters, Milk Amyloid A, Somatic Cell Count, and Some Pathogens in Buffalo Milks

Being resistant to hard environmental conditions and diseases makes Buffalo to have its valuable milk. Microbial contamination may occur due to undesirable conditions such as mastitis, environmental contamination, and stress. If microorganisms are not removed from the milk, it causes many production disadvantages including inadequacy of production, failure of fermentation and shortening of the shelf life. This study was conducted to determine the relationship between somatic cells count (SCC), the presence of some pathogens, and milk amyloid A (MAA) in the buffalo milk. In addition, oxidative stress in buffalo milk was evaluated. For this purpose, 70 samples were collected and Enterobacteriaceae, coliform microorganisms, Escherichia coli, Salmonella spp. analyses were performed. Biochemical parameters [Malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), catalase, superoxide dismutase (SOD), and total antioxidant capacity (TAC)] and serological parameter (MAA) were measured. The SCC was not statistically different according to Enterobacteriaceae, coliform microorganisms, E. coli groups. While MDA, NO, SOD, and TAC values were not statistically different according to the SCC groups, GSH and catalase values were statistically different. MAA values were statistically significant compared to the SCC groups. Moreover, there was a positive correlation between MAA and MDA/SCC. Detection of MAA may prevent the mixing of healthy and mastitic milk. Therefore, more reliable buffalo milk products will be presented to consumption.

Acute Phase Protein as Biomarker for Diagnosis of Sub-Clinical Mastitis in Cross-Bred Cows

The present study was carried out to investigate the role of acute phase proteins [Serum amyloid A (SAA) and milk amyloid A (MAA)] for diagnosis of sub-clinical mastitis in cross-bred cows. The study was conducted on 40 apparently healthy cross-bred cows, in the mid-lactation period and were equally divided into four groups [Group-I (Bacteriologically negative milk samples, California mastitis test (CMT) negative), Group-II (Bacteriologically negative with SCC £200,000 cells/ml milk, CMT positive), Group-III (Bacteriologically negative with SCC ³200,000 £500,000 cells/ml milk, CMT positive) and Group-IV (Bacteriologically positive with SCC ³200,000 £500,000 cells/ml milk, CMT positive)]. On bacteriological examination of 57 milk samples, 27(47.36%) were found positive and 30 (52.63%) were found negative. A highly significant (P 200,000 cells/ml, 74 µg/ml and 10 µg/ml, respectively for the diagnosis of sub-clinical mastitis with high sensitivity (90% to 100%). The area under curve of the SAA was larger than those of SCC and MAA, which suggests that the SAA test was more accurate than SCC and MAA for the diagnosis of sub-clinical mastitis.

Milk amyloid A as a biomarker for diagnosis of subclinical mastitis in cattle.

Background and Aim:Mastitis is one of the most vital noteworthy monetary risks to dairy ranchers and affects reproductive performance in dairy cattle. However, subclinical mastitis (SCM) negatively affects milk quality and quantity and associated with economic losses as clinical mastitis. It is recognizable only by additional testing. Somatic cell count (SCC) is currently used worldwide for the screening of intramammary infection (IMI) infections. However, somatic cells (SC) are affected by numerous factors and not always correlate with infection of the udder. Therefore, the aim of the present study was to evaluate the milk amyloid A (MAA) in the milk of normal and SCM cows and compare the sensitivity of both MAA secretion and SCC in response to mammary gland bacterial infection.Materials and Methods:A total of 272 quarter milk samples collected from 68 Friesian cows after clinical examination for detection of clinical mastitis were employed in this study. All quarter milk samples (272) were subjected to bacteriological examination, while SCs were assessed in samples (220). Following SCC estimation and bacteriological examination, the apparently normal quarter milk samples were categorized into 7 groups and MAA concentration was estimated in normal and subclinical mastitic milk samples.Results:Prevalence of clinical mastitis was 19.12 % (52 quarters), while 80.88 % (220 quarters) were clinically healthy with normal milk secretion. Of those 220 clinically healthy quarter milk samples, 72 (32.73%) showed SCM as detected by SCC (SCC ≥500,000 cells/ml). The most prevalent bacteria detected in this study were streptococci (48.53%), Staphylococcus aureus (29.41%), Escherichia coli (36.76%), and coagulase-negative staphylococci (11.76%). Results of MAA estimation revealed a strong correlation between MAA secretion level and SCC in agreement with the bacteriological examination. Interestingly, there was a prompt increase in MAA concentration in Group III (G III) (group of milk samples had SCC ≤200,000 cells/ml and bacteriologically positive) than Group I (G I) (group of milk samples with SCC ≤500,000 cells/ml and bacteriologically negative), as MAA concentration in G III was about 4 times its concentration in G I.Conclusion:Our study provides a strong evidence for the significance of MAA measurement in milk during SCM, and MAA is more sensitive to IMI than SCC. This can be attributed to rapid and sensitive marker of inflammation. The advantage of MAA over other diagnostic markers of SCM is attributed the minute or even undetectable level of MAA in the milk of healthy animals, it is not influenced by factors other than mastitis, and could be estimated in preserved samples. Therefore, we recommend that estimation of MAA concentration in milk is a more useful diagnostic tool than SCC to detect SCM and to monitor the udder health in dairy cattle.

Study on the accuracy of milk amyloid A test and other diagnostic methods for identification of milk quality.

Bulk tank somatic cell count (BTSCC) is a gold standard test for identification of milk quality, but its results are influenced by several interventional factors. Recently, application of acute phase proteins and especially milk amyloid A (MMA) has been considered as accurate parameters for milk quality study. The current research was done to evaluate the accuracy of MMA, BTSCC, fat, protein and lactose for identification of milk quality. Ninety bulk tank milk samples were collected from 30 randomly selected dairy herds and classified into two groups of samples with BTSSC > 200000 cells per mL and those with BTSSC < 200000 cells per mL. Protein, fat, lactose and MAA contents of samples were analyzed. Average amount of the MAA in healthy and mastitic milk samples were 5.15 and 504.35 ng mL-1, respectively. Statistically significant difference was seen for MAA and total protein contents between two groups. Clinical accuracy of MAA-, total protein-, fat- and lactose–based methods was 0.937, 0.757, 0.665 and 0.547, respectively. The MAA method at concentration of 20.78 ng mL-1 had the highest sensitivity (97.30%) and specificity (46.70%). Evaluation of MAA is recommended as a rapid and accurate method for determination of the unfavorable changes in milk quality and early subclinical mastitis diagnosis.

Test characteristics of milk amyloid A ELISA, somatic cell count, and bacteriological culture for detection of intramammary pathogens that cause subclinical mastitis.

Bovine mastitis is an important disease in the dairy industry, causing economic losses as a result of withheld milk and treatment costs. Several studies have suggested milk amyloid A (MAA) as a promising biomarker in the diagnosis of mastitis. In the absence of a gold standard for diagnosis of subclinical mastitis, we estimated the diagnostic test accuracy of a commercial MAA-ELISA, somatic cell count (SCC), and bacteriological culture using Bayesian latent class modeling. We divided intramammary infections into 2 classes: those caused by major pathogens (e.g., Escherichia coli, Staphylococcus aureus, streptococci, and lacto-/enterococci) and those caused by all pathogens (major pathogens plus Corynebacterium bovis, coagulase-negative staphylococci, Bacillus spp., Streptomyces spp.). We applied the 3 diagnostic tests to all samples. Of 433 composite milk samples included in this study, 275 (63.5%) contained at least 1 colony of any bacterial species; of those, 56 contained major pathogens and 219 contained minor pathogens. The remaining 158 samples (36.5%) were sterile. We determined 2 different thresholds for the MAA-ELISA using Bayesian latent class modeling: 3.9 µg/mL to detect mastitis caused by major pathogens and 1.6 µg/mL to detect mastitis caused by all pathogens. The optimal SCC threshold for identification of subclinical mastitis was 150,000 cells/mL; this threshold led to higher specificity (Sp) than 100,000 cells/mL. Test accuracy for major-pathogen intramammary infections was as follows: SCC, sensitivity (Se) 92.6% and Sp 72.9%; MAA-ELISA, Se 81.4% and Sp 93.4%; bacteriological culture, Se 23.8% and Sp 95.2%. Test accuracy for all-pathogen intramammary infections was as follows: SCC, sensitivity 90.3% and Sp 71.8%; MAA-ELISA, Se 88.0% and Sp 65.2%; bacteriological culture, Se 83.8% and Sp 54.8%. We suggest the use of SCC and MAA-ELISA as a combined screening procedure for situations such as a Staphylococcus aureus control program. With Bayesian latent class analysis, we were able to identify a more differentiated use of the 3 diagnostic tools. The MAA-ELISA is a valuable addition to existing tools for the diagnosis of subclinical mastitis.

Milk amyloid a—a potential biomarker to detect subclinical mastitis in lactating dromedary camel

The objective of the present study was to investigate the possible use of Milk Amyloid A (MAA) as a sensitive biomarker to detect subclinical mastitis in the lactating dromedary camel. Quarter milk samples (n=120), from 65 milking dromedary camels, were collected to evaluate somatic cell count (SCC) and MAA. At the area under the curve of 0.859 for MAA (P<0.001) and cut off points of 306000 cells/ml and 1040 ng/ml for SCC and MAA, respectively. The sensitivity and specificity of the MAA test to detect subclinical mastitis were 100 and 43.9%, respectively. In conclusion, it might be possible to use MAA measurement, as screening test, for early detection of sub-clinical mastitis in dromedary camel.

Expression of acute phase proteins and inflammatory cytokines in mouse mammary gland following Staphylococcus aureus challenge and in response to milk accumulation.

We used a mouse model of pathogenic (Staphylococcus aureus) and non-pathogenic (teat sealing) mammary inflammation to investigate mRNA expression of several inflammatory cytokines and acute phase proteins (APP) in mammary tissue and liver, and the appearance of some of these factors in plasma and milk. The expression levels of IL1β and TNFα were markedly up-regulated in Staph. aureus-inoculated mammary tissue at 72h, whilst IL6 was up-regulated to a lesser extent in a way which was not confined to the inoculated glands. APP expression was up-regulated at 48 and 72h in both Staph. aureus-inoculated and teat-sealed mammary glands. These differences between cytokine and APP expression provide additional support for the contention that APPs are produced within the mammary tissue itself during inflammation, rather than in associated immune cells. We propose that measurement of cytokines and APP in combination might provide a tool for diagnostic discrimination between mastitis caused by pathogenic invasion and milk accumulation, and hence allow for better targeting of antibiotic therapy. In comparison with mammary expression, expression of cytokines in liver tissue was up-regulated to a similar or lesser extent, whilst expression of APP was up-regulated to a much greater extent. The first appearance of increased cytokine and APP concentrations in plasma and of milk amyloid A (MAA) in milk occurred in advance of the measurable up-regulation of expression, hence their origin cannot be stated with certainty.

Production of recombinant proteins in microalgae at pilot greenhouse scale

Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae.

Milk haptoglobin, milk amyloid A, and N-acetyl-β-d-glucosaminidase activity in bovines with naturally occurring clinical mastitis diagnosed with a quantitative PCR test

The associations between quantitative bacteriological results from a real-time PCR test and concentrations of acute-phase proteins (APP) and N-acetyl-β-d-glucosaminidase (NAGase) activity in milk in naturally occurring clinical mastitis were investigated. Milk APP concentrations and NAGase activity in clinical mastitis caused by different udder pathogens were studied. The associations between the severity of the clinical signs and concentrations of APP and NAGase activity were estimated. Milk samples from 281 cases of clinical mastitis were collected from 3 Estonian dairy farms and analyzed by PCR to identify pathogens. Twenty-seven samples out of 281 (9.6%) were PCR negative. Milk samples containing 4 or more bacterial species (n=28) were considered possibly contaminated and excluded from all further analyses. In total, 443 bacterial identifications were made from the remaining 226 milk samples. A single bacterial species was detected in 68 samples (30.1%), 2 species were detected in 99 samples (43.8%), and 3 species were detected in 59 (26.1%) samples. To determine the inflammatory response in the udder, the concentrations of milk amyloid A (MAA) and haptoglobin (Hp) and NAGase activity in the milk were analyzed. A significant positive association was found between the severity of the clinical signs and inflammatory markers in the milk. Milk amyloid A and Hp concentrations and NAGase activity were significantly higher in samples with large quantities of bacterial DNA from Escherichia coli or Streptococcus dysgalactiae compared with milk samples not containing those species. Large quantities of bacterial DNA from Trueperella pyogenes or Streptococcus uberis in the milk were associated with elevated concentrations of Hp and high NAGase activity, but not with increased MAA concentrations. Milk samples containing Corynebacterium bovis and coagulase-negative staphylococci had significantly lower concentrations of MAA and Hp and lower NAGase activity compared with samples where these species were not detected. It can be concluded that concentrations of APP and NAGase activity in the milk were associated with the quantity of bacterial DNA in the milk samples.

Effects of combined liver and udder biopsying on the acute phase response of dairy cows with experimentally induced E. coli mastitis

A minimally invasive biopsy technique was evaluated for udder tissue collection in dairy cows with Escherichia coli mastitis. Meanwhile, the effect of taking repeated liver and udder biopsies on the systemic and local acute phase response (APR) of the dairy cows was investigated during the disease. The cows were divided into a biopsy group (B) (n = 16) and a no-biopsy group (NB) (n = 16) and were sampled in the acute disease stage and in the recovery stage. The cows’ pre-disease period served as a control period for establishing baseline values for the investigated parameters. A total of 32 Holstein-Friesian cows were inoculated with 20 to 40 colony-forming units (cfu) of E. coli in one front quarter at 0 hour. Liver biopsies were collected at −144, 12, 24 and 192 h, and udder biopsies were collected at 24 and 192 h post E. coli inoculation (PI) using a minimally invasive biopsy technique. Effects of combined biopsying were investigated by recording production traits, clinical response, and measuring inflammatory milk and blood parameters: E. coli, somatic cell count, milk amyloid A (MAA) levels, white blood cell count, polymorphonuclear neutrophilic leukocyte numbers and serum amyloid A levels at several time points. E. coli inoculation changed all production parameters and the clinical and inflammatory response in all cows except one that was not infected. Combined biopsying had no constant or transient effect on the daily feed intake, the clinical responsiveness or the blood parameters, but affected the daily milk yield and some milk parameters transiently, that is, the presence of blood in milk, increased E. coli counts and MAA levels during the acute disease stage. Combined biopsying had no effect on the parameters in the recovery stage apart from the presence of blood in the milk. In conclusion, although, a minimally invasive biopsy technique was used, tissue damages could not be avoided when biopsying and they transiently affected the inflammatory parameters in the mammary gland. Nevertheless, we believe combined biopsying of liver and udder is as an acceptable approach to study the systemic and local APR in dairy cows during E. coli mastitis, if the timing of biopsying and other types of sampling is planned accordingly.

Diagnostic importance of the concentration of milk amyloid A in quarter milk samples from dairy cows with mastitis

Acute phase proteins have been used as biomarkers of inflammation. Their concentrations increase in milk from cows with latent and subclinical mastitis. The aim of our study was to evaluate milk amyloid A (MAA) as indicator of udder inflammation. We used 24 dairy cows from a herd with 120 Slovak Pied cattle. In addition to bacteriological examination, the following indicators were determined in all quarter milk samples. On the basis of results of clinical examination, Californian mastitis test (CMT), and number of Somatic cell count (SCC), four groups of quarter milk samples were formed. The levels of MAA in both subgroups of Group 1 (healthy cows), divided by the number of SCC - IA (n = 10), IB (n = 15), determined at repeated samplings, differed significantly from the initial levels (P &lt; 0.01). Highly significant correlation (R2 = 0.272), was detected between SCC, and MAA in Group 2 (n = 27) at individual collections (P &lt; 0.01), while in the Group 3 (n = 25) the level of both indicators decreased by the end of the experiment despite higher initial concentrations (P &lt; 0.01). No significant differences in MAA were observed between individual collections in Group 4 (n = 6). No positive correlation (R2 = 0.525) was detected between SCC and MAA in this group. The obtained results allowed us to conclude that MAA in milk can act as a marker of inflammation of the udder only in the initial, asymptomatic stages of dairy cow mastitis. The experiment was one of first studies with MAA in Slovak Pied cattle.

The diagnostic value of determination of positive and negative acute phase proteins in milk from dairy cows with subclinical mastitis

Currently, somatic cell count (SCC) and bacterial culture are considered as the gold standard methods for the diagnosis of bovine subclinical mastitis. However, SCC has a low diagnostic accuracy. Therefore, for identification of infected animals, new biomarkers with high diagnostic accuracy are needed. Acute phase proteins (APPs) are proteins that are increased (positive APPs) or decreased (negative APPs) in response to inflammation. The objective of this study was to determine the diagnostic value of milk APPs for the diagnosis of subclinical mastitis in dairy cows. A total of 90 clinically healthy cows were randomly selected. Of these, 52 cows were considered subclinical mastitic based on a SCC higher than 130 × 1,000 cells/mL of milk and positive bacterial culture results of milk samples obtained from at least one of the quarters. Milk amyloid A (MAA) concentration was measured using a commercial ELISA kit and albumin, α-lactalbumin, β-lactoglobulin, and immunoglobulin (Ig) were measured in the whey samples by the use of cellulose acetate electrophoresis. Diagnostic sensitivity and specificity and cutoff points for each test were determined via receiver-operating characteristics analysis. Significant (P 1.6 mg/L. The results of this study showed that determination of MAA can be used as a reliable method for the diagnosis of bovine subclinical mastitis.

Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis

Coagulase-negative staphylococci (CNS) are in several countries the most common bacteria isolated in subclinical mastitis. To investigate the innate immune response of cows to infections with two common mastitis-causing CNS species, Staphylococcus epidermidis and Staphylococcus simulans, experimental intramammary infection was induced in eight cows using a crossover design. The milk somatic cell count (SCC), N-acetyl-β-D-glucosaminidase (NAGase) activity, milk amyloid A (MAA), serum amyloid A (SAA) and proinflammatory cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor α (TNF-α) were determined at several time points before and after challenge. All cows became infected and showed mild to moderate clinical signs of mastitis. The spontaneous elimination rate of the 16 infections was 31.3%, with no difference between species. Infections triggered a local cytokine response in the experimental udder quarters, but cytokines were not detected in the uninfected control quarters or in systemic circulation. The innate local immune response for S. simulans was slightly stronger, with significantly higher concentrations of IL-1β and IL-8. The IL-8 response could be divided into early, delayed, or combined types of response. The CNS species or persistency of infection was not associated with the type of IL-8 response. No significant differences were seen between spontaneously eliminated or persistent infections.

Enzyme activity and acute phase proteins in milk utilized as indicators of acute clinical E. coli LPS-induced mastitis

The importance of non-visual and on-line monitoring of udder health increases as the contact between humans and animals decreases, for example, in robotic milking systems. Several indicator systems have been introduced commercially, and a number of techniques are currently in use. This study describes the kinetics of seven indigenous milk parameters for monitoring udder inflammation in an Escherichia coli lipopolysaccharide (LPS, endotoxin)-induced mastitis model. Proportional milk from LPS-infused quarters was compared with milk from parallel quarters, which were placebo-treated with sterile 0.9% NaCl solution. Somatic cell counts (SCCs), the acute phase proteins (APP), that is, milk amyloid A (MAA) and haptoglobin (Hp), and the enzymes N-acetyl-β-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), alkaline phosphatase (AP) and acid phosphatase (AcP) were measured at fixed intervals during the period from −2 to +5 days after LPS and NaCl infusions. All parameters responded significantly faster and were more pronounced to the LPS infusions compared with the NaCl infusions. All parameters were elevated in the proportional milk collected at the first milking 7 h after infusion and developed a monophasic response, except Hp and MAA that developed biphasic response. SCC, LDH, NAGase and Hp peaked at 21 h followed by AP, AcP and MAA peaking at 31 h with the highest fold changes seen for MAA (23 780×), LDH (126×), NAGase (50×) and Hp (16×). In the recovery phase, AP, AcP and Hp reached base levels first, at 117 h, whereas LDH, NAGase and MAA remained elevated following the pattern of SCC. Minor increases of the milk parameters were also seen in the neighboring (healthy) quarters. Distinction between inflamed and healthy quarters was possible for all the parameters, but only for a limited time frame for AP and AcP. Hence, when tested in an LPS mastitis model, the enzymes LDH, NAGase and AP in several aspects performed equally with SCC and APP as inflammatory milk indicators of mastitis. Furthermore, these enzymes appear potent in the assessment of a valuable time sequence of inflammation, a necessary ingredient in modeling of programs in in-line surveillance systems.

Evaluation of milk haptoglobin and amyloid A in high producing dairy cattle with clinical and subclinical mastitis in Shiraz

Mastitis is one of the most common diseases in dairy cattle and results in considerable loss of animals. This study was designed to evaluate milk haptoglobin (Hp) and milk amyloid A (MAA) as an inflammatory indicator for clinical and subclinical mastitis of cattle in dairy farms in Shiraz, Iran. Forty-three subclinical mastitic cows with a positive California Mastitis Test (CMT) and no clinical signs of mastitis, 28 clinical mastitic cows, and 10 healthy cows with negative CMT were selected. After confirmation of clinical and subclinical mastitis by bacterial identification, milk samples were taken from four quarters of each cow and mixed, and one sample was taken from the pooled milk. The most dominant isolated bacterium from clinical and subclinical samples was Staphylococcus aureus (n = 25; 35.2%). The most dominant isolated bacterium from clinical (19/28) and subclinical (11/43) samples was Staphylococcus spp. Of isolated bacteria of milk in cattle with clinical mastitis, 67.8% (n = 19) was S. aureus. There was no bacterial growth in 37.1% (n = 16) of cattle with subclinical mastitis. Of isolated bacteria of milk in cattle with subclinical mastitis, 13.9% (n = 6) and 11.6% (n = 5) was S. aureus and Staphylococcus epidermidis, respectively. There were significant differences (P 13.43 μg/ml for MAA, >9.71 ng/ml for milk Hp, and >14 × 104 cell per millilitre for somatic cell count with corresponding 100% sensitivity and 100% specificity for MAA, 83.72% sensitivity and 100% specificity for milk Hp, and 88.37% sensitivity and 100% specificity for somatic cell count. The results of this study reveal that MAA is a sensitive factor for diagnosis of subclinical mastitis in cattle.

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein–Friesian cows were challenged with S. chromogenes 4 weeks after calving . One udder quarter of each cow was inoculated with 2.1 × 10 6 cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-β- d-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12 h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on–off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.