Mammary Associated Serum Amyloid A3 Research Articles

321 ASAS-EAAP Talk: Quantitative proteomics in animal and veterinary science: a pipeline for exploiting advanced analytical technology

Abstract Application of proteomic investigation to veterinary and animal sciences has grown over the last decade, but has still not reached its full potential of application in animal health and production research [1]. Nevertheless, establishing a versatile methodology has allowed the application of quantitative proteomics for increased understanding of physiological and pathophysiological challenges, and especially to identify potential biomarkers of disease in a range of animal species. A pipeline of sample preparation and mass spectrometry followed by statistical, bioinformatic and biochemical analyses has been established to deal with biofluids and tissue samples from cattle, sheep, pigs, chickens, dogs and cats as well as wild animals. Quantitative proteomic investigation of milk in an experimental model of Streptococcus uberis mastitis of dairy cows has identified potential novel biomarkers with implications for diagnosis and treatment of this major disease. Proteins in milk which have potential as disease biomarkers, such as cathelicidin, haptoglobin and mammary associated serum amyloid A3, are significantly increased in abundance during bovine mastitis. Proteomic investigation has confirmed that these biomarkers are also increased in milk during subclinical and clinical mastitis. Proteomic analysis of plasma from chicken following stimulation of the inflammatory response to Escherichia coli lipopolysaccharide endotoxin has characterised major changes in the chicken plasma proteome. Novel biomarker candidates of hemopexin and fatty acid binding protein have been identified. This proteomic pipeline can be incorporated into many areas of research, providing novel findings at the forefront of animal and veterinary science. Such proteomic investigation requires close interdisciplinary collaboration between experts in mass spectrometry, bioinformatics, statistics and animal production in order to fully exploit recent technological advances in the omic sciences.[1] P. Bilic, et al, Proteomics in Veterinary Medicine and Animal Science: Neglected Scientific Opportunities with Immediate Impact, Proteomics. 47 (2018) 1–7. doi:10.1002/pmic.201800047.

A pilot study of acute phase proteins as indicators of bovine mastitis caused by different pathogens

This study analysed three acute phase proteins in milk from natural cases of bovine mastitis and compared their profiles across different pathogens causing the infection. Their ability to differentiate subclinical and clinical mastitis from normal (uninfected) milk samples was also examined. Samples from various dairy farms across Scotland submitted to the Veterinary Diagnostic Services unit of the University of Glasgow were used for this study. They were subjected to microbiological examination for mastitis pathogens, evaluation of somatic cell counts and analyses by ELISAs for haptoglobin, C-reactive protein and mammary associated serum amyloid A3. Each acute phase protein (APP) was compared across pathogens and form of mastitis. Significant differences (P = 0.000) were observed for each APP between causative pathogen and form of mastitis. There were significant correlations between the pathogen and the form of mastitis and the 3 APP showed similar profile for the different pathogen type and forms of mastitis. It can be concluded that the aetiological pathogen of mastitis to a large extent influences the clinical form of the disease, this, ultimately being reflected in the degree and course of secretions of the acute phase proteins; Hp, M-SAA3 and CRP into milk during mastitis. Variations of which, show correspondent patterns with related pathogen/form-of-mastitis.

Early post parturient changes in milk acute phase proteins.

The periparturient period is one of the most critical periods in the productive life of a dairy cow, and is the period when dairy cows are most susceptible to developing new intramammary infections (IMI) leading to mastitis. Acute phase proteins (APP) such as haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) have been detected in milk during mastitis but their presence in colostrum and milk in the immediate postpartum period has had limited investigation. The hypothesis was tested that APP are a constituent of colostrum and milk during this period. Enzyme linked immunosorbent assays (ELISAs) were used to determine each APP's concentration in colostrum and milk collected daily from the first to tenth day following calving in 22 Holstein-Friesian dairy cows. Haptoglobin was assessed in individual quarters and composite milk samples while M-SAA3 and CRP concentration were determined in composite milk samples. Change in Hp in relation to the high abundance proteins during the transition from colostrum to milk were evaluated by 1 and 2 dimension electrophoresis and western blot. In 80% of the cows all APPs were detected in colostrum on the first day following parturition at moderately high levels but gradually decreased to minimal values in the milk by the 6th day after calving. The remaining cows (20%) showed different patterns in the daily milk APP concentrations and when an elevated level is detected could reflect the presence of IMI. Demonstration that APP are present in colostrum and milk following parturition but fall to low levels within 4 days means that elevated APP after this time could be biomarkers of post parturient mastitis allowing early intervention to reduce disease on dairy farms.

Effect of pre-analytical treatments on bovine milk acute phase proteins.

BackgroundSamples for diagnostic procedures often require some form of pre-analytical preparation for preservation or safe handling during transportation prior to analysis in the laboratory. This is particularly important for milk samples which frequently need preservatives to retain milk composition as close to that found in freshly collected samples as possible.MethodsMilk samples were treated by heating at 56 °C for 30 min or preserved by addition of either potassium dichromate or bronopol respectively. Haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were measured in the various treatment groups and in control samples which were not treated, using enzyme linked immunoassays. The concentrations of each APP were compared between treated and non-treated groups using the Wilcoxon signed ranks tests.ResultsHeat treatment of samples was found to have a significant lowering effect on milk M-SAA3 and CRP but not Hp. The use of potassium dichromate and bronopol as preservatives in milk had no significant effects on milk Hp and M-SAA3 concentration but lowered milk CRP values compared to controls.ConclusionsThe observed effects of heating and preservative use on milk APP should be taken into consideration when assaying samples which have undergone heat treatment as a result of international transfer regulations involving biological samples or samples needing chemical preservation prior to transport to laboratory.

The major acute phase proteins of bovine milk in a commercial dairy herd.

BackgroundMilk acute phase proteins (APP) have been identified and show promise as biomarkers of mastitis. However analysis of their profile in dairy cows from a production herd is necessary in order to confirm their benefits in mastitis diagnosis. The profiles of milk haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were determined in 54 composite milk (milk from all functional quarters of a cow’s udder collected in a common receptacle) samples (CMS) from a commercial dairy farm. Milk Hp was also determined in individual quarter milk (milk from a single udder quarter) samples (QMS) (n = 149) of the cows.An ELISA was developed and validated for the determination of milk Hp while commercial kits were used for M-SAA3 and CRP assay respectively. Composite milk APP results were compared with cow factors including parity, stage of lactation, percentage protein and fat as well as somatic cell counts (SCC).ResultsComposite milk Hp ranged from <0.4–55 μg/ml with a median of 3.5 μg/ml; composite milk M-SAA3 ranged from <0.6–50 μg/ml and had a median of 1.2 μg/ml, while CRP ranged from <1.80–173 ng/ml and had a median of 24.6 ng/ml. Significant correlations were found between composite SCC and Hp (P-value <0.009) as well as parity and Hp (P < 0.009), but not between M-SAA3 and SCC, M-SAA3 and Hp, M-SAA3 and CRP or M-SAA3 and parity. Milk CRP was correlated with % fat (P = 0.002) and % protein (P = 0.001) of the milk samples. The lack of correlation of SCC with the M-SAA3 and CRP could result from these APP being more sensitive to intra-mammary infection than SCC. Quarter milk Hp had a range of <0.4–420 μg/ml with a median value of 3.6 μg/ml, with 92 % of samples below 20 μg/ml.ConclusionBaseline values of Hp, M-SAA3 and CRP were established in composite milk from cows with normal SCC on the dairy farm. Parity was recognized as a possible confounding factor when diagnosing mastitis using Hp. The value of the APP, Hp, M-SAA3 and CRP as substitutes or to complement SCC in indicating udder inflammation, was demonstrated.

Enhanced Membrane-tethered Mucin 3 (MUC3) Expression by a Tetrameric Branched Peptide with a Conserved TFLK Motif Inhibits Bacteria Adherence*

We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a transcription factor protein/DNA array. MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides -242 and -62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection.

Acute phase proteins: Biomarkers of infection and inflammation in veterinary medicine

Acute phase proteins (APPs) have been used as biomarkers of inflammation, infection and trauma for decades in human medicine but have been relatively under-utilised in the context of veterinary medicine. However, significant progress has been made in the detection, measurement and application of APPs as biomarkers in both companion and farm animal medicine over recent years. In the dog, C-reactive protein, haptoglobin and serum amyloid A have been identified as significant diagnostic 'markers' of steroid-responsive meningitis-arteritis, while in cats and cattle haptoglobin and alpha(1) acid glycoprotein and haptoglobin and serum amyloid A have proved valuable biomarkers of disease, respectively. In dairy cattle, haptoglobin and a mammary-associated serum amyloid A3 isoform, produced by the inflamed mammary gland during episodes of mastitis, have great potential as biomarkers of this economically important disease. Understanding the use of APP as biomarkers of inflammatory conditions of domestic animals has expanded significantly over recent years, and, with the insights provided by ongoing research, it is likely that these compounds will be increasingly used in the future in the diagnosis and prognosis of both companion and farm animal disease.

Evaluation of colostrum-derived human mammary-associated serum amyloid A3 (M-SAA3) protein and peptide derivatives for the prevention of enteric infection:in vitroand in murine models of intestinal disease

In vitro experiments confirmed that a 10-mer peptide derived from human mammary-associated serum amyloid A3 (M-SAA3) protected intestinal epithelial cells from enteropathogenic Escherichia coli (EPEC) adherence. The entire 42-mer human M-SAA3 protein was even more effective, reducing EPEC binding by 72% relative to untreated cells (P<0.05), compared with 25% and 57% reductions for the human 10-mer and Lactobacillus GG, respectively. However, none of the M-SAA3 peptides reduced Salmonella invasion in vitro (P>0.05). Each of the M-SAA3 10-mer peptides and the 42-mer was then administered orally to mice at 500 mug day(-1) for 4 days before deliberate infection with either Citrobacter rodentium (mouse model of EPEC) or Salmonella Typhimurium. None of the peptides protected against Salmonella infection and the 42-mer may even increase infection, as there was a trend towards increased Salmonella counts in the liver and small intestine in 42-mer-treated mice compared with those in sodium acetate-treated control mice. Citrobacter counts were reduced in the caecum of mice administered the 42-mer relative to a scrambled 10-mer (P<0.05), but not compared with the sodium acetate control and no reductions were observed in the faeces or colon. Overall, although promising anti-infective activity was demonstrated in vitro, neither the 42-mer M-SAA3 protein nor a 10-mer peptide derivative prevented enteric infection in the animal models tested.

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis.

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.

Induction of human mammary-associated serum amyloid A3 expression by prolactin or lipopolysaccharide

In most mammalian species, serum amyloid A isoform 3 (SAA3) appears to be the predominant SAA isoform expressed extrahepatically. However, human SAA3 gene expression has not been detected previously and, therefore, this gene was referred to as a pseudogene. We report for the first time the transcriptional expression of human SAA3. Human SAA3 gene expression was detected by RT-PCR after stimulation of mammary gland epithelial cells with either prolactin (PRL) or lipopolysaccharide (LPS). The full-length 655 bp cDNA sequence for this mammary-associated serum amyloid A3 ( M-SAA3) was obtained using 5 ′ and 3 ′ rapid amplification of cDNA ends (RACE). The human M-SAA3 transcript would conceptually translate into a 42 residue mature protein, which is smaller than other mammalian SAA3 isoforms that are typically 104–113 amino acids in length. This study defines the cDNA sequence for human SAA3 and also demonstrates the upregulation of M-SAA3 expression in response to the lactational hormone PRL or to an acute phase stimulant such as LPS.

The Conserved TFLK Motif of Mammary-Associated Serum Amyloid A3 Is Responsible for Up-regulation of Intestinal MUC3 Mucin Expression In Vitro

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.

The conserved TFLK motif of mammary-associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in vitro.

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.

Human serum amyloid A3 peptide enhances intestinal MUC3 expression and inhibits EPEC adherence

We previously determined that the N-terminal region of bovine mammary-associated serum amyloid A3 (M-SAA3) increased intestinal mucin MUC3 levels in HT29 human intestinal cells by ∼2.5-fold, relative to untreated cells. This study shows that the human M-SAA3 N-terminal peptide further enhances MUC3 transcript levels by ∼4.3-fold in these cells ( p<0.02), implicating a species-specific interaction. Furthermore, immunofluorescence and immunoblot analysis using a MUC3-specific monoclonal antibody confirms that the human M-SAA3 peptide stimulates MUC3 protein expression and secretion by the HT29 cells. More importantly, pretreatment of the cells with the peptide causes a subsequent 73% decrease in the adherence of enteropathogenic Escherichia coli (EPEC) to these cells, relative to untreated cells ( p<0.01). The intestinal mucin MUC3 has been shown to provide a protective barrier in the gut and inhibit adherence of pathogens to the gut wall. Therefore, a means to increase MUC3 protein expression by a colostrum-associated peptide or protein may be a highly effective prophylactic treatment for the prevention of gastrointestinal diseases such as necrotizing enterocolitis and infectious diarrhea.

Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A 3 (M-SAA3) into colostrum

Mammary-associated serum amyloid A 3 (M-SAA3) was secreted at highly elevated levels in bovine, equine and ovine colostrum and found at lower levels in milk 4 days postparturition. N-terminal sequencing of the mature M-SAA3 protein from all the three species revealed a conserved four amino acid motif (TFLK) within the first eight residues. This motif has not been reported to be present in any of the hepatically-produced acute phase SAA (A-SAA) isoforms. Cloning of the bovine M-Saa3 cDNA from mammary gland epithelial cells revealed an open reading frame that encoded a precursor protein of 131 amino acids which included an 18 amino acid signal peptide. The predicted 113 residue mature M-SAA3 protein had a theoretical molecular mass of 12,826 Da that corresponded with the observed 12.8 kDa molecular mass obtained for M-SAA3 in immunoblot analysis. The high abundance of this extrahepatically produced SAA3 isoform in the colostrum of healthy animals suggests that M-SAA3 may play an important functional role associated with newborn adaptation to extrauterine life and possibly mammary tissue remodeling.