Distinguishing Natural Infections of the Bovine Mammary Gland by Staphylococcus from Streptococcus spp. Using Quantitative Milk Proteomics.

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Lycopene abolishes palmitate-mediated myocardial inflammation in female Wistar rats via modulation of lipid metabolism, NF-κB signalling pathway, and augmenting the antioxidant systems.

BACKGROUND AND AIMS: Obesity-related heart failure is exacerbated by excessive intake of saturated fats such as palmitate (PA). Lycopene (LYC) possesses anti-lipidemic, antioxidant, cytoprotective, and anti-inflammatory effects. This study, therefore, evaluated the impact of LYC against PA-invoked cardiotoxicity. METHODS AND RESULTS: Thirty-six female rats were equally divided into six groups: control; PA (5 mM); PA + LYC (24 mg/kg); PA + LYC (48 mg/kg); LYC (24 mg/kg); and LYC (48 mg/kg). The PA was administered five times weekly for seven weeks, while the LYC was given for the last two weeks. Lipids in the blood and the heart were estimated, as were oxidative stress and antioxidant indices, cardiac function, inflammation, and histology. Palmitate overload occasioned a significant (p < 0.05) increase in cardiac cholesterol (50%), phospholipids (19%), and non-esterified fatty acids (40%). However, triglyceride levels decreased (38%). Furthermore, malondialdehyde (45%), hydrogen peroxide (33%) levels and myeloperoxidase activity increased (79%). Also, cardiac gamma-glutamyl transferase (50%), serum creatine kinase activities (1.34 folds), NF-kB, interleukin1ß, and interleukin-6 mRNA expression increased in the PA group relative to the control. In contrast, reduced glutathione (13%) and nitric oxide levels (22%), interleukin-10 mRNA expression, cardiac creatine kinase (35%), lactate dehydrogenase (33%), aspartate, and alanine transaminase activities decreased markedly (15- and 10%, respectively). Also, PA caused hyperemia, congestion of the cardiac interstitium, and infiltration of inflammatory cells. However, treatment with LYC reversed the features of cardiotoxicity and histological complications caused by PA. These observations are likely because LYC has anti-inflammatory, antioxidant, and cytoprotective properties. CONCLUSION: Thus, LYC might be an appropriate remedy to manage PA-induced cardiotoxicity in female rats.

Inhibition of fat accumulation, lipid dysmetabolism, cardiac inflammation, and improved nitric oxide signalling mediate the protective effects of lycopene against cardio-metabolic disorder in obese female rats.

Obesity, hallmarked by excessive lipid accumulation and dysregulation, continues to escalate the prevalence of cardiometabolic diseases, and is a foremost cause of deaths globally. Alternative therapeutic agents are urgently needed. This study hypothesized that lycopene could proffer beneficial effects against obesity-induced cardiometabolic changes. Obesity was induced using a Western-style diet. Female albino rats (n = 36) were randomized into 6 groups of 6 rats each: normal control, obese control, obese + lycopene (20 mg/kg body weight [b.wt.]), obese + lycopene (40 mg/kg b.wt.), lycopene (20 mg/kg b.wt.), and lycopene (40 mg/kg b.wt.). The study was 10 weeks. Obese rats had significantly higher (P< .05) body weight and total body fat. Lipids (triacylglycerol, cholesterol [CHOL], and free fatty acids), cardiac injury markers (troponin-T, creatine kinase-myocardial band, and malondialdehyde), and cardiovascular risk markers (low-density lipoprotein-CHOL, atherogenic and coronary risk indices) were significantly (P< .05) elevated in obese rats compared with control groups. However, obesity significantly reduced high-density lipoprotein-CHOL and impaired cardiac nitric oxide signalling. Pro-inflammatory mediators (nuclear factor-κB-p65, interleukin-1ß [IL-1ß], and IL-6) transcripts were increased in the heart of obese rats, whereas cardiac IL-10 expression was repressed. Treatment with lycopene reduced lipid concentrations, normalized lipid and lipoprotein metabolism, augmented nitric oxide concentration and IL-10 messenger RNA transcripts, and attenuated the expression of pro-inflammatory mediators. These findings delineate the role of lycopene in the attenuation of cardiometabolic disorders potentiated by obesity.

Lycopene suppresses palmitic acid-induced brain oxidative stress, hyperactivity of some neuro-signalling enzymes, and inflammation in female Wistar rat.

Neuroinflammation can be triggered by certain high caloric nutrients such as palmitic acid (PA). The effect of lycopene against PA-induced neuroinflammation in female rats has not been as explored. In the present study, thirty rats (weighing 150-200) g were randomly allotted into six groups (n = 5) comprising normal control, PA control, PA + lycopene (0.24 mg/kg), PA + lycopene (0.48 mg/kg), lycopene (0.24 mg/kg), and lycopene (0.48 mg/kg), respectively. After seven weeks of PA challenge (5 mM) including two weeks of lycopene treatment, the brain was excised for analyses. Palmitic acid overload caused significant (p < 0.05) increases in adenosine deaminase, monoamine oxidase-A, nucleotides tri-phosphatase, 5'-nucleotidase, acetylcholine esterase, and myeloperoxidase activities, and malondialdehyde (MDA) levels which were reduced significantly in the lycopene-treated groups. Conversely, catalase and glutathione peroxidase activities, and reduced glutathione levels concentration decreased by 43%, 34%, and 12%, respectively in the PA control groups compared with the Control. Also, PA triggered a decrease in the brain phospholipids (11.43%) and cholesterol (11.11%), but increased triacylglycerol level (50%). Furthermore, upregulated expressions of Interleukin-1ß, Interleukin-6, and NF-ĸB-p65 in the PA control were attenuated, while decreased Interleukine-10 expression was upregulated due to lycopene treatment. Severe brain vacuolation observed in the histology of the PA control rats was normalized by lycopene. This study concludes that lycopene ameliorated PA-induced neuroinflammation, probably via attenuation of oxidative stress, and downregulation of TLR4/ NF-κB -p65 axis.

Lycopene abrogates obesity-provoked hyperactivity of neurosignalling enzymes, oxidative stress and hypothalamic inflammation in female Wistar rats.

Obesity, a global epidemic, has been strongly associated with impairment of brain function. Lycopene has several therapeutic properties and can cross the blood-brain barrier. However, its effects on obesity-provoked brain dysfunction remain unexplored. This study evaluated the potential remediating effects of lycopene on obesity-induced neurological derangements. Thirty-six female Wistar rats (150-200g) were distributed in six groups (n = 6); normal control, obese control, obese + lycopene (20 mg/kg), obese + lycopene (40 mg/kg), normal + lycopene (20 mg/kg), and normal + lycopene (40 mg/kg). Obesity was induced by feeding rats with the Western diet for eight weeks, while normal rats received the control diet. Afterwards, the brain was excised and processed for biochemical, gene expression analyses, and histological evaluations. Obesity-induced brain dysfunction was hallmarked by reduced brain organosomatic index, accumulation of lipids in the cerebrum, and hyperactivity of neurotransmitters-metabolizing enzymes (AChE, ADA, MAO-A, 5'-nucleotidase, and NTPdase). Also, obese rats had decreased antioxidant capacity, with increased oxidative damage, while the expressions of NF-κß p65 and pro-inflammatory cytokines (IL-1ß and IL-6) were elevated in the hypothalamus. These observations were validated by histomorphological evaluations, which showed vacuolation in the brain of obese rats. Treatment with lycopene significantly (p < 0.05) reduced the elevated lipid contents and activities of neuronal enzymes, alleviated oxidative stress and inflammation, while improving the histology of the brain, in a dose-dependent manner. Thus, lycopene abrogates obesity-provoked brain dysfunction and may present a safe and viable therapeutic option for the management of neurological perturbations associated with obesity.

Quantitative distribution and interaction of Salmonella Zega with host cells in visceral organs of chickens infected orally, intraperitoneally and per cloaca.

Immunohistochemical study of the visceral organs of chickens experimentally infected with Salmonella Zega by three routes was carried out to compare the quantitative distribution and interaction of the organism with host cells. 100 birds comprising of 2 week-old chickens were divided into 4 groups of 25 each. Group A was inoculated orally, group B intraperitoneally, group C were administered per cloaca and D were not inoculated and served as control. All the infected birds were inoculated with 0.2 ml of 1 × 108 cfu of the bacteria. Two birds from each group were sacrificed every 24 h post infection. Samples of visceral organs were collected for immunohistochemistry. The distribution of Salmonella Zega in every organ was taken as Mean ± SD of the number of foci of immunoreactions and Compared using a 2-way ANOVA. The interaction of Salmonella Zega with host cells was determined by taking the percentage of the days post infection in which immunoreactions were detected in host cells in each route of infection. The distribution of the organism was highest in the lung of intraperitoneally infected chickens (83.95 ± 27.89) and lowest in the heart (5.21 ± 3.65) of chickens that were infected per cloaca. The highest percentage of interaction of Salmonella Zega was recorded in the epithelial (100%) and blood (100%) cells in all the routes of infection. There were variations in the distribution of Salmonella Zega in visceral organs of chickens but the level of interactions with host cells were similar even when infected through different routes.

Acute phase proteins in bitches subjected to conventional and minimally invasive ovariohysterectomy / Proteínas de fase aguda em cadelas submetidas à ovariohisterectomia convencional e minimamente invasiva

The aim of this study was to evaluate and to compare the possible inflammatory changes by screening acute phase proteins concentrations in healthy bitches subjected to ovariohysterectomy. Minimally invasive and conventional (laparotomy) ovariohysterectomies were performed in 17 client-owned adult female mixed breed dogs. Nine animals were subjected to minimally invasive and eight animals to conventional ovariohysterectomy. Blood samples were taken before surgery, 24, 48 hours, and seven days postoperatively. Serum C-reactive concentration was determined by a commercial ELISA kit and serum haptoglobin concentration was measured via hemoglobin binding assay, both previously validated for use in dogs. As the data did not meet the normal distribution criteria, the nonparametric Kruskall-Wallis was performed to compare quantitative variables between groups. One-way ANOVA and the Friedman test were used for multiple comparisons between time points, with a P<0.05 considered significant. C-reactive protein concentration was significantly different (P<0.0001) at 24 hours postoperatively between groups. There was no significant difference in haptoglobin concentration between groups. C-reactive protein and haptoglobin concentrations were significantly different at 24 and 48 hours postoperatively for minimally invasive and conventional ovariohisterectomies. These findings provided an overview of the short-term inflammatory effects produced by minimally invasive and conventional ovariohysterectomies.(AU)

O objetivo deste trabalho foi avaliar e comparar o efeito inflamatório, por meio da determinação de proteínas de fase aguda, produzido por dois protocolos cirúrgicos distintos de ovariohisterectomia em cadelas. Para tanto, foram determinadas as concentrações de proteína C reativa (CRP) e haptoglobina (Hp) de 17 cadelas, sem raça definida, adultas, sendo nove animais submetidos à ovariohisterectomia pela técnica convencional por laparotomia e oito a ovariohisterectomia pela técnica minimamente invasiva. Para avaliar a resposta de fase aguda causada pelo trauma cirúrgico, amostras de sangue foram obtidas antes dos procedimentos cirúrgicos e em quatro momentos distintos após as cirurgias (24, 48 horas e sete dias) para as dosagens de CRP e Hp, com kit comercial ELISA e via ensaio de ligação com a hemoglobina, respectivamente, validados para o uso em cães. Como não houve distribuição normal dos dados, utilizou-se o teste de Kruskall-Wallis para comparação das variáveis quantitativas entre os dois grupos. Os testes de Friedman e One-way ANOVA foram usados para comparações múltiplas entre os momentos avaliados, sendo considerado P<0,05 como significante. Houve diferença significativa (P<0,0001) para as concentrações de CRP 24 horas após o procedimento cirúrgico entre os dois protocolos utilizados. Não houve diferença significativa para as concentrações de Hp entre os dois protocolos cirúrgicos. Foram evidenciadas alterações significativas para as concentrações de CRP e Hp 24 e 48 horas após as ovariohisterectomias, independentemente da técnica cirúrgica utilizada. As concentrações de CRP e Hp demonstraram o efeito inflamatório imediato induzido pela ovariohisterectomia convencional e minimamente invasiva.(AU)

Blood serum acute phase proteins and iron dynamics during acute phase response of Salmonella enterica serotype Dublin experimentally infected buffalo calves.

The study aimed to evaluate clinical signs, blood serum acute phase proteins (APP) and iron dynamics during the acute phase response (APR) of Salmonella Dublin experimentally infected Murrah buffalo calves. Six buffalo calves constituted the control group (CNT) and six were orally inoculate with 108 CFU of S. Dublin (INF). Clinical evaluation was performed, rectal swabs to detect S. Dublin strains were collected and venous blood was sampled before and throughout seven days after inoculation. The APP fractions ß-haptoglobin, α-haptoglobin, ceruloplasmin and transferrin were analyzed by 1-D and 2-D electrophoresis. Proteins were identified using LC/ESI-MS/MS and NCBI database. Plasma fibrinogen, serum iron and serum haptoglobin concentrations were measured. The inoculation of 108 CFU of S. Dublin was effective in inducing clinical signs of Salmonellosis, such as hyperthermia and diarrhea. 1-DE showed that ß and α-haptoglobin increased 204% (p = 0.008) and 184% (p = 0.022) 48 h after inoculation (HAI), respectively, with highest concentrations 120 HAI (498% increased, p = 0.012; 431% increased, p = 0.011) and 168 HAI (492% increased, p = 0.019; 523% increased, p = 0.028). 2-DE showed that the expression of two spots, identified as ß-haptoglobin, were increased 693% (p = 0.0006) and 580% (p = 0.0003) 168 HAI, respectively, while one spot, identified as α-haptoglobin, increased 714% (p = 0.040). Haptoglobin concentrations increased 1339% (p < 0.0001) 168 HAI. 1-DE showed that ceruloplasmin increased 42% (p = 0.034) 48 HAI, with highest concentration 120 HAI (133% increased, p = 0.022). 2-DE showed that the expression of two spots, identified as ceruloplasmin, were increased 218% (p = 0.0153) and 85% (p = 0.0143) 168 HAI, respectively. Fibrinogen increased 78% (p = 0.012) 96 HAI, with highest concentration 120 HAI (increased 114%, p = 0.002). Iron decreased 33% 24 HAI (p = 0.015) and 37% 72 HAI (p = 0.029), and began to be restored 96 HAI. 1-DE showed that transferrin decreased 23% 120 HAI (p = 0.047), and that values were restored 168 HAI. 2-DE showed that expression patterns of transferrin comparing 0 h and 168 HAI were similar, evidencing that values were restored 168 HAI. In conclusion, the inoculation of 108 CFU was effective in inducing hyperthermia and diahrrea. ß and α-haptoglobin, ceruloplasmin and fibrinogen worked as positive APP during the APR to S. Dublin infection and are potential biomarker candidates. Concentrations of iron and transferrin decreased during the infection, highlighting the fact that mechanisms for restricting iron availability are part of the APR triggered against S. Dublin infection in buffalo calves.

Reference 1D and 2D electrophoresis maps for potential disease related proteins in milk whey from lactating buffaloes and blood serum from buffalo calves (Water buffalo, Bubalus bubalis).

The aim of this study was to identify potential disease related proteins in milk whey of lactating buffaloes and blood serum of buffalo calves, in order to define a reference electrophoresis map for 1-DE and 2-DE. Additionally, changes in some protein patterns from buffalo calves during salmonellosis and lactating buffaloes during mastitis are presented. Milk samples were collected and distributed into groups: Milk samples from healthy buffaloes (SCC < 100.000 cells/ml, negative microbiology and CMT) (G1, n = 5) and buffaloes with subclinical mastitis (SCC > 500.000 cells/ml, positive microbiology and CMT) (G2, n = 5). Blood samples from buffalo calves (n = 6) were collected, and three calves were experimentally infected with Salmonella Dublin and samples analyzed before (M0) and 72 h after inoculation (M1). 1-DE was accomplished by loading 10 µg of TP into SDS-PAGE, stained with Coomassie blue. 2-DE was accomplished by loading 200 µg of TP into 11 cm, pH 3-10 non-linear IPG strips, followed by SDS-PAGE, stained with Coomassie blue. Protein bands/spots were excised, subjected to tryptic in-gel digestion and analyzed by LC/ESI-MS/MS. Protein identity was assigned using NCBI databases. After bands/spots from 1-DE and 2-DE were analyzed, a protein map with 35 and 40 different identified proteins in blood serum and milk whey, respectively, was generated. Significant changes in patterns of haptoglobin were observed in buffalo calves with salmonellosis and in patterns of IgLC, ß-lactoglobulin and α-lactalbumin of lactating buffaloes during mastitis. The establishment of a protein map for 1-DE and 2-DE, identifying potential disease related proteins, can help to address alterations during diseases in buffaloes.

Acute phase proteins in dogs naturally infected with the Giant Kidney Worm (Dioctophyme renale).

BACKGROUND: Dioctophyme renale is a nematode parasite of dogs, usually found in the right kidney, causing severe damage to the renal parenchyma. OBJECTIVES: The objective was to evaluate the acute phase response in dogs naturally infected with this Giant Kidney Worm and the possible effects of nephrectomy on circulating concentrations of select acute phase proteins (APP) such as serum amyloid A (SAA), C-reactive protein (CRP), and haptoglobin (HP). METHODS: Nephrectomy was performed in infected dogs and the worms were collected for identification. Blood samples were taken 24 hours before surgery, and 4, 8, and 12 hours postoperatively on the following 10 consecutive days, and 28 days after surgery. Acute phase protein concentrations were determined at all time points. Cortisol concentrations were determined 24 hours before surgery and at recovery (28 days after surgery). One-way ANOVA and Friedman test were used for multiple comparisons; the Wilcoxon-signed rank test was used to compare variables, and Spearman's rho rank test was used to assess the correlation between the number of parasites recovered from the dogs and the APP concentration. RESULTS: Forty-five parasites were recovered from the 12 dogs evaluated in this study. Dogs showed significantly increased HP concentrations (P < .05) but lower CRP and SAA concentrations before surgery, and cortisol concentrations were significantly higher at admission when compared to recovery. No significant correlations were found between the number of parasites and APP concentrations. CONCLUSION: There is a particular acute phase response profile in dogs with kidney worm infection. Nephrectomy induced a short-term inflammatory process.

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.

BACKGROUND: Samples 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. METHODS: Milk 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. RESULTS: Heat 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. CONCLUSIONS: The 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.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 1. High abundance proteins, acute phase proteins and peptidomics.

A peptidomic investigation of milk from an experimental model of Streptococcus uberis mastitis in dairy cows has incorporated a study of milk high abundance and acute phase (APP) proteins as well as analysis of low molecular weight peptide biomarkers. Intramammary infection (IMI) with S. uberis caused a shift in abundance from caseins, ß-lactoglobulin and α-lactalbumin to albumin, lactoferrin and IgG with the increase in lactoferrin occurring last. The APP response of haptoglobin, mammary associated serum amyloid A3 and C-reactive protein occurred between 30-48 hours post challenge with peak concentrations of APPs at 72-96 hours post challenge and declined thereafter at a rate resembling the fall in bacterial count rather than the somatic cell count. A peptide biomarker panel for IMI based on capillary electrophoresis and mass spectrometry was developed. It comprised 77 identified peptides (IMI77) composed mainly of casein derived peptides but also including peptides of glycosylation dependent cell adhesion molecule and serum amyloid A. The panel had a biomarker classification score that increased from 36 hour to 81 hour post challenge, significantly differentiating infected from non-infected milk, thus suggesting potential as a peptide biomarker panel of bovine mastitis and specifically that of S. uberis origin. The use of omic technology has shown a multifactorial cross system reaction in high and low abundance proteins and their peptide derivatives with changes of over a thousand fold in analyte levels in response to S. uberis infection.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 3. Untargeted metabolomics.

Intramammary infection leading to bovine mastitis is the leading disease problem affecting dairy cows and has marked effects on the milk produced by infected udder quarters. An experimental model of Streptococcus uberis mastitis has previously been investigated for clinical, immunological and pathophysiological alteration in milk, and has been the subject of peptidomic and quantitative proteomic investigation. The same sample set has now been investigated with a metabolomics approach using liquid chromatography and mass spectrometry. The analysis revealed over 3000 chromatographic peaks, of which 690 were putatively annotated with a metabolite. Hierarchical clustering analysis and principal component analysis demonstrated that metabolite changes due to S. uberis infection were maximal at 81 hours post challenge with metabolites in the milk from the resolution phase at 312 hours post challenge being closest to the pre-challenge samples. Metabolic pathway analysis revealed that the majority of the metabolites mapped to carbohydrate and nucleotide metabolism show a decreasing trend in concentration up to 81 hours post-challenge whereas an increasing trend was found in lipid metabolites and di-, tri- and tetra-peptides up to the same time point. The increase in these peptides coincides with an increase in larger peptides found in the previous peptidomic analysis and is likely to be due to protease degradation of milk proteins. Components of bile acid metabolism, linked to the FXR pathway regulating inflammation, were also increased. Metabolomic analysis of the response in milk during mastitis provides an essential component to the full understanding of the mammary gland's response to infection.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 2. Label-free relative quantitative proteomics.

Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously.

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

BACKGROUND: Milk 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). RESULTS: Composite 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. CONCLUSION: Baseline 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.