Milk Amyloid 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.

Novel Approach for Evaluating Pregnancy-Associated Glycoprotein and Inflammation Markers during the Postpartum Period in Holstein Friesian Cows.

We evaluated the relationship between decreased pregnancy-associated glycoprotein (PAG) levels, inflammatory parameters (serum amyloid A [SAA] and milk amyloid A [MAA]), postpartum inflammatory conditions (mastitis, ketosis, and follicular cysts), and the FOXP3 gene. Nineteen Holstein-Friesian cows were included in this study. Up to approximately eight weeks after delivery, weekly health examinations were performed for mastitis and ketosis, and reproductive organ ultrasonography was performed. The decreasing PAG rate was negatively correlated with SAA concentration (r = -0.493, p = 0.032). Cows with mastitis exhibited a slower trend of PAG decrease (p = 0.095), and a greater percentage of these cows had MAA concentrations above 12 µg/mL (p = 0.074) compared with those without mastitis. A negative correlation, although nonsignificant (r = -0.263, p = 0.385), was observed between the day-open period and decreased PAG rate. The day-open period was correlated with the presence or absence of follicular cysts (p = 0.046). Four cows that developed follicular cysts were homozygous for the G allele of the FOXP3 gene related to repeat breeders. These results indicate a relationship between a decreased PAG rate and inflammatory status during the postpartum period. Thus, suppressing inflammation during the perinatal period may improve reproductive efficiency in the dairy industry.

Siyah Alaca, Kırmızı Alaca ve Simental sığır sütlerinde somatik hücre sayısı ile süt amiloid A, elektriksel iletkenlik ve pH arasındaki ilişkiler

Bu çalışmada Siyah Alaca, Kırmızı Alaca ve Simental sığır sütlerinde somatik hücre sayısı (SHS) ile süt amiloid A (SAA), elektiksel iletkenlik (Eİ) ve pH arasındaki ilişkilerin araştırılması amaçlanmıştır. Siyah Alaca (46 baş) ve Kırmızı Alaca (13 baş) sığırlarının % 87 ve % 78’i, Simental sığırlarının (82 baş) ise hiçbiri SHS bakımından risk grubunda bulunmamıştır. Çalışmada somatik hücre sayısı Siyah Alaca, Kırmızı Alaca ve Simental ırklarında sırasıyla 4.49±0.079, 4.40±0.091 ve 4.20±0.111 hücre ml-1 olarak belirlenmiştir. SAA ortalama düzeyleri Siyah Alaca ırkında 343.80±26.40, Kırmızı Alaca ırkında 286.20±24.520, Simental ırkında 479.85±76.79 ngml-1 olarak hesaplanmıştır. Süt örneklerinin pH değerleri Siyah Alaca ırkında ise 6.62±0.017, Kırmızı Alaca ırkında 6.67±0.016, Simental ırkında 6.58±0.084 tespit edilmiştir. Eİ değerleri Siyah Alaca ırkında 3.58±0.050, Kırmızı Alaca ırkında 3.65±0.071, Simental ırkında 1.21±0.019 mS cm-1 olarak ölçülmüştür. Elde edilen sonuçlar, Siyah alaca ve Kırmızı Alaca ırklarında SHS ile SAA arasında anlamlı bir yakın ilişkinin düşük konsantrasyonlarda bile farkedilebiliyor olması süte özgü SAA'nın subklinik mastitisin potansiyel bir fizyolojik belirteci olduğunu düşündürebilir. EI ve pH ölçüm sonuçlarının SHS ve SAA ile anlamlı ilişki içinde olmaması, bu belirteçlerin hastalık tespitinden ziyade süt kalitesinin belirlenmesinde kullanılabileceğini göstermiştir.

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).

Reliability of udder infrared thermography as a non-invasive technology for early detection of sub-clinical mastitis in Sahiwal (Bos indicus) cows under semi-intensive production system

The present study focused on Sahiwal cows, a prominent milch breed in tropical India, to correlate udder temperature with physiological markers of stress and inflammation during subclinical mastitis (SCM). The primary goal was to assess the potential of udder infrared thermography for the early detection of SCM under the semi-intensive production. Cows were categorized based on milk somatic cell counts (SCC), with healthy (H) cows having SCC <2 × 105 cells/mL and no history of mastitis, and cows with subclinical mastitis (SCM) and initial stages of clinical mastitis (CM) having quarter milk SCC of 2–5 × 105 and >5 × 105 cells/mL, respectively. Firstly, udder thermograms were analysed for udder skin surface temperature (USST), teat skin surface temperature (TSST), and teat apex temperature (TAT) using Fluke software to determine the optimal site for temperature measurement during intramammary infection. Secondly, milk samples were collected for automatic estimation of compositional changes, electrical conductivity, and pH. Thirdly, milk whey was separated for quantifying stress and inflammatory indicators, including cortisol, prolactin, and acute-phase proteins (APPs): milk amyloid A and milk haptoglobin using bovine-specific ELISA kits. Significant increases (p < 0.01) in USST, TSST, TAT, cortisol, and APPs were observed in SCM and CM compared to healthy cows, while prolactin levels decreased (p < 0.01). The correlation matrix revealed strong positive correlations of SCC with USST (r = 0.84, p < 0.01). In ROC analysis, USST demonstrated cut-off values of 37.74 and 39.58 °C, with accuracy (p < 0.05) of 98% for SCM and 95% for CM, surpassing both TAT and TSST. Therefore, the combination of these non-invasive methods increases the reliability and accuracy of infrared thermography for early detection of SCM, providing valuable insights for the development of a protocol for routine screening and udder health monitoring in indigenous dairy cows.

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.

Using behavioral observations in freestalls and at milking to improve pain detection in dairy cows after lipopolysaccharide-induced clinical mastitis

This study aimed to determine the effect of lipopolysaccharide (LPS)-induced mastitis with or without nonsteroidal anti-inflammatory drug (NSAID) on dairy cows' clinical, physiological, and behavioral responses in the milking parlor and freestalls as well as the specificity (Sp) and sensitivity (Se) of behavioral responses in detecting cows with LPS-induced mastitis. Twenty-seven cows received an intramammary infusion of 25 µg of Escherichia coli LPS in 1 healthy quarter. Following LPS infusion, 14 cows received a placebo (LPS cows), and 13 cows received 3 mg/kg of body weight of ketoprofen i.m. (LPS+NSAID cows). Cow response to the challenge was monitored at regular intervals from 24 h before to 48 h postinfusion (hpi) through direct clinical observations, markers of inflammation in milk, and via point-in-time direct behavioral observations in the barn and at milking. In LPS cows, infusion induced a significant increase of plasma cortisol levels at 3 and 8 hpi, milk cortisol levels at 8 hpi, somatic cell counts from 8 to 48 hpi, IL-6 and IL-8 at 8 hpi, milk amyloid A (mAA) and haptoglobin at 8 and 24 hpi, rectal temperature at 8 hpi, and respiratory rate at 8 hpi. Their rumen motility rate decreased at 8 and 32 hpi. Compared with before the challenge, significantly more LPS cows stopped feeding/ruminating and pressed their tail between their legs at 3 and 5 hpi, increased feeding/ruminating at 24 hpi, and had the tendency to be less responsive, dropping their head, and dropping their ears at 5 hpi. At milking, compared with before challenge, significantly more LPS cows lifted their hooves at forestripping at 8 hpi. The 2 groups showed similar patterns of response for milk cortisol, somatic cell count, respiratory rate, mAA, haptoglobin, and IL-6, IL-1β, and IL-8. Compared with LPS cows, LPS+NSAID cows had significantly lower plasma cortisol levels at 3 hpi, their rectal temperature decreased at 8 hpi, their rumen motility rate increased at 8 and 32 hpi, and their heart rate increased at 32 hpi. Compared with LPS cows, a significantly larger proportion of LPS+NSAID cows were feeding/ruminating, a lower proportion had ears down at 5 hpi, and a larger proportion lied down at 24 hpi. At milking, whatever the phase of milking, for "hoof to belly," 9 out of 14 cows did not show this behavior before infusion (Sp = 64%) and 14/14 did not kick during pre-infusion milking (Sp = 100%). Regarding sensitivity, at maximum, 5 cows out of 14 (Se = 36%) displayed "hoof to belly" after infusion. For "lifting hoof," 14/14 did not show hoof-lifting before infusion (Sp = 100%) and 6/14 displayed it after infusion (Se = 43%) at forestripping only. In the freestall barn, 9 behaviors had a Sp >75% (at minimum, 10/14 did not show the behavior) whatever the time point but Se < 60% (at maximum, 8/14 displayed the behavior). Finally, "absence of feeding and ruminating" had Sp of 86% (12/14 ate/ruminated) and Se of 71% (10/14 did not eat/ruminate) at 5 hpi. This study shows that feeding/ruminating, tail position, and reactivity at forestripping could be used as behavioral indictors for early detection of mastitis-related pain in dairy cows.

Lactate dehydrogenase: Detecting high bacterial and somatic cells counts in goats from whole milk samples

The efficacy of using only somatic cell count (SCC) in goats to study the milk quality as a single test has been widely questioned, with total bacterial count being considered as ancillary test in several studies. Therefore, the main objective of this study was to identify new biomarkers of interest that correlate with high SCC and TBC values in milk samples. The concentration of five selected proteins according to their association with infected and injured udders (milk amyloid A, lactoferrin, pentraxin 3, N-acetyl-beta-d-glucosaminidase and lactate dehydrogenase) were determined in bulk raw milk together with SCC and TBC values. Only LDH showed a significant positive correlation with respect to SCC or TBC, no correlation between the other proteins with respect to both parameters were detected. Following, LDH activity and SCC and TBC values were analyzed in a wide diversity of goat milk samples performing a receiver operating characteristic (ROC) curve analysis. LDH showed a significant positive correlation with both SCC and TBC values. LDH activity differed significantly between milk samples with a high and/or low SCC and TBC with a sensitivity that can reach values of 91.3 % and a specificity of 83.2 %. Our results, highlight LDH activity measurement as a biomarker of interest in raw milk for the detection of low-quality milk samples which could potentially be used for the diagnosis of subclinical mastitis in goats.

Investigation of the Relationship Between Somatic Cell Count and Milk Amyloid A, Electrical Conductivity and pH in Cow Milk

Mastitis, üretim kaybı, meme hasarı ve buna bağlı tedavi maliyetleri açısından süt endüstrisi için büyük bir maliyettir. Somatik hücre sayısı (SHS), mastitin bir göstergesi olarak yaygın olarak kullanılmaktadır. Bu çalışmada inek sütlerinde süt amiloid A (SAA), elektriksel iletkenlik (Eİ) ve pH gibi biyokimyasal göstergelerin SHS ile karşılaştırılması amaçlanmıştır. Afyon ili inek sütü üreticilerinden toplamda 50 adet inek sütü toplanmıştır. Toplanan sütler SHS’na göre 200 bin hücre mL-1 altında olan Grup I (GI) ve 200-400 bin hücre mL-1 arasında olan Grup II (GII) ve 400 bin hücre/mL üstünde olanlar ise Grup III (GIII) olmak üzere 3 gruba ayrılmıştır. Toplanan sütlerin SHS gruplarına göre SAA değerlerinin istatistiksel olarak anlamlı farklılık gösterdiği ve SHS değerleri arttıkça SAA medyan değerlerinin de arttığı gözlenmiştir (p

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.

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.

Facile construction of a molecularly imprinted polymer-based electrochemical sensor for the detection of milk amyloid A.

Amolecularly imprinted electrochemical sensor for the detection of serum amyloid A (MAA) in milk was established for early diagnosis of subclinical mastitis in dairy cows. The electrochemical sensor was initially constructed using a nanocomposite material (reduced graphene oxide/gold nanoparticles, AuNPs@rGO) to modify the working electrode. The template protein, MAA, was then immobilized using pyrrole as the functional monomer to carry out the electropolymerization. Finally, the template protein was removed to form a molecular imprint film with the capability to qualitatively and quantitatively signaling of MAA. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and scanning electron microscopy (SEM) were used to characterize the modification process of themolecularly imprinted electrochemical sensors. Under optimized conditions, the sensor shows two well-behaved linear relationships in the MAA concentration range 0.01 to 200ng/mL. A lower detection limit was estimated to be 5pg/mL (S/N= 3). Other parameters including the selectivity, reproducibility (RSD 3.2%), and recovery rate (96.1-103%) are all satisfactory. Compared with the traditional methods, detection of MAA to determine the subclinical mastitis of dairy cows can efficiently bediagnosed and henceprevent an outbreak of dairy cow mastitis. The electrochemical sensor can detect MAA more rapidly, sensitively, and inexpensively than the ELISA-based MAA detection. These advantages indicate that the method is promising for early diagnosis of dairy cows.

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.

Evaluation of Intramammary Platelet Concentrate Efficacy as a Subclinical Mastitis Treatment in Dairy Cows Based on Somatic Cell Count and Milk Amyloid A Levels

Evaluation of Intramammary Platelet Concentrate Efficacy as a Subclinical Mastitis Treatment in Dairy Cows Based on Somatic Cell Count and Milk Amyloid A Levels

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.

Innate immunity and metabolomic responses in dairy cows challenged intramammarily with lipopolysaccharide after subacute ruminal acidosis

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in dairy cows known to elicit local and systemic immune responses. We recently showed that cows experiencing SARA and challenged intramammarily with lipopolysaccharide (LPS) experienced stronger metabolic disturbances compared with cows without SARA. Therefore, we hypothesized that cows experiencing SARA have a modulated innate immune response and impaired plasma metabolome compared with healthy cows when experiencing an acute mastitis challenge. A total of 18 Simmental cows were subjected either to a Control (CON, n=6) or SARA (n=12) feeding regimen, receiving either 40% or 60% concentrates for 30 days. Thereafter, six SARA (SARA-LPS) and the CON (CON-LPS) cows were intramammarily challenged with 50 µg LPS from Escherichia coli (O26 : B6), while the remaining six SARA cows (SARA-PLA) received a placebo. Blood and milk samples were analyzed for acute phase proteins and a targeted ESI-LC-MS/MS-based metabolomics approach was performed in blood samples 24 h after the LPS challenge. The LPS infusion caused a strong increase in immune response variables, with a higher concentration of milk amyloid A 48 h after the LPS challenge in SARA-LPS compared with CON-LPS cows. Cows receiving the LPS infusion had a lower plasma concentration of several amino acids and lysophosphatidylcholines but without differences in SARA cows and healthy cows. In conclusion, our results revealed that an intramammary LPS infusion increased acute phase proteins and modulated the blood metabolome. While no systemic differences between SARA and healthy cows were observed, cows experiencing SARA showed a higher concentration of an acute phase protein at the local level of the mammary gland. Further research is required to elucidate the underlying mechanisms and to evaluate its clinical significance for udder health.

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.

Colostrum and milk protein rankings and ratios of importance to neonatal calf health using a proteomics approach

Administration of colostrum to the newborn calf before gut closure is pivotal to its health, because of the transfer of passive immunity. Traditionally, passive immunity has been attributed to the transfer of immunoglobulins although it is increasingly clear that multiple other factors contribute, including innate immune proteins, developmental factors, immunomodulatory factors, and the presence of cellular immunity. The objective of this study was to produce a comprehensive comparison of the bovine colostrum proteome and the milk proteome by applying 2-dimensional liquid chromatography-tandem mass spectrometry. Further, the objectives were to rank proteins mutually and generate protein ratios from the spectral counts of the 2 proteomes and ELISA to gain insight into which proteins could be of most relevance to neonatal calf health. To obtain an in-depth picture of the bovine colostrum and milk proteome, we compared the contents of different fractions from bovine colostrum and milk from our 2 previous studies. A total of 140 colostrum fluid-phase proteins and 103 milk fluid-phase proteins were detected. In the cellular fraction, 324 and 310 proteins were detected in colostrum and milk, respectively. In total, 514 proteins were detected, of which 162 were in the fluid phase. Of these, 50 proteins were exclusively seen in colostrum, 13 were exclusively seen in milk, and 99 were common to colostrum and milk. Ranking of proteins mutually and calculating protein ratios based on spectral counts and ELISA resulted in new information on how proteins were associated with the fluid or cellular fraction of the samples. Interestingly, despite lower counts/concentrations than the classical proteins such as immunoglobulins, β-lactoglobulin, and lactotransferrin, several proteins appeared in higher or similar colostrum:milk spectral count ratios as these. Using this approach indicated, for example, that osteopontin, haptoglobin, milk amyloid A, and gelsolin may be interesting molecules to study in detail in their relation to calf health. Although the sensitivity, identification, and ranking of proteins varied between the 2 methods, and proteome analysis clearly suffers from low sensitivity, we believe that this idea and approach of generating ratios and ranking proteins can contribute new information and perspectives on how to prioritize the importance of multiple proteins, beyond immunoglobulins, in relation to neonatal calf health.