Acute phase proteins, proteomics and metabolomics in the diagnosis of bovine mastitis

Bovine mastitis continues to pose a major economic challenge to the dairy industry worldwide. Critical to the management and control of this condition, is the need for prompt and accurate diagnosis in field conditions, therefore a search for more sensitive and reliable biomarkers is required. In this thesis, studies focused on assessing milk samples from cows with various forms of mastitis were undertaken with a view to identifying new biomarkers for bovine mastitis. Three acute phase proteins (APP); haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were measured in milk samples from composite milk samples of all lactating cows in a commercial dairy herd, mastitis cases, submitted to a diagnostic laboratory and following an experimental mastitis challenge of cows with Streptococcus uberis. A new enzyme linked immunosorbent assay (ELISA) was developed for measuring Hp, while commercial ELISA assay kits were used to assay M-SAA3 and CRP. Other mastitis related parameters evaluated in the samples included the somatic cell counts (SCC) and the presence of pathogens. A reliable and sensitive ELISA was developed and optimized for measuring milk Hp. A cut off value for Hp of 7.9 μg/ml was established for milk with SCC less than 200,000 cells/ml. Pathogen-specific variations were observed in the concentration of each APP in mastitic milk. It was observed that the environmental pathogens showed higher concentrations of APP compared to other pathogens, from the study of mastitis milk samples submitted to the diagnostic laboratory. Also, it was possible to distinguish between samples from subclinical and clinical mastitis and between samples from subclinical and healthy udders using each of the APP (P<0.05). Haptoglobin, M-SAA3 and CRP showed corresponding variation with stage of infection during the course of experimental mastitis, and specifically CRP was observed to rise earlier than other two APP. Furthermore, characterization of the profile of these APP in the immediate post-calving milk samples was carried out to determine how valuable they would be in recognizing new mastitis infections arising at the post-partum period. It was observed that there is a general moderately-high level of APP in milk immediately following parturition which drops a few days later in healthy milk. The immunohistochemical localization of Hp in the bovine mammary gland was also assessed. It could be concluded from that study that neutrophils and the mammary epithelial cells secrete Hp into milk during mastitis. Gel and non-gel based proteomics approaches were employed to study the protein profiles and variation in mastitic milk from normal samples. Several proteins were identified that confirmed previous findings and project new mastitis markers, for example, serotransferrin, serpins, alpha-macroglobulin and neutrophil gelatinase associated lipocalins. A capillary electrophoresis mass spectrometry system (CE-MS) was also employed to elucidate the changing peptidome in milk during the course of an experimental mastitis, which lead to the generation of a panel of 77 polypeptides, which were able to significantly differentiate critical stages of mastitis. Three of these polypeptides were found in mastitic milk samples from previous peptidomic analyses thereby indicating strong biomarker value. Finally, a liquid chromatography mass spectrometry based metabolomics approach was used to study the changing profile of small metabolites in milk during the course of an experimental infection. Several pathway-based changes that highlighted metabolites of potential significance in mastitis diagnosis were recognized including lactose synthesis, nitrogen containing compounds such as betaine, L-carnitine and lipid metabolites pathways namely sn-glycerophosphocholine and choline among others. Overall, this study has shown the value of APP, milk proteomics and metabolomics in bovine mastitis diagnosis; the changing proteins and metabolites or their patterns need to be further experimentally and clinically validated as specific and sensitive markers of mastitis. Ultimately, the applicability of APP, proteins, peptides and metabolites and/or their changing patterns as mastitis biomarkers would require their adaptation to rapid (on farm) and robust measurement formats

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

Review of studies published on the medicinal importance of different parts of Citrullus lanatus in the last ten years

Watermelon (Citrullus lanatus), (CL) is an edible fruit of Cucurbitaceae family. It is cultivated worldwide for it is nutritive and medicinal values. The records of online scientific publications on CL were accessed using Google, Google Scholar, PubMed, science.gov, Scopus, and Worldwide science as search engines, were collected from January 2010 to April 2021 and analyzed using descriptive statis tics. Emphasis was placed on phytochemical, proximate, antioxidant, and pharmacological published articles on different parts of CL during this period. A total of 121 published articles that focused on different parts of CL in the last ten years were retrieved with phytochemicals 17.4% (21), proximate 7.4% (9), antioxidants 6.6% (8), pharmacology 68.6% (83). The pharmacology field was subdivided into antimicrobial 14.9% (18), cardioprotective 10.2% (13), reproduction 9.2% (12), toxicology and hepatoprotective 6.6% (8) each, analgesic, and anti-inflammatory 5.8%,  europrotective 3.3% (4), anthelmintics (0.8%). Considering publications on different parts of CL, the seed received the highest attention with 42.1% (51) followed by fruits 35.5% (43), rind 18.1% (22), leaf 2.5% (3) while the least was whole fruit 1.7% (2). It was observed in this review on published articles that the CL fruits received the highest level of attention considering the phytochemicals, proximate, and antioxidant components to exhibit good antimicrobial potentials. While the CL leaf received little attention on antimicrobial ability. Also, different parts exhibited cardioprotective, reproduction, toxicology, hepatoprotective, neuroprotective, analgesic, and anti-inflammatory activities, anti-ulcerative efficacy due to phytochemicals, antioxidant, and proximate constituents in different parts of CL. It is worth noting that neuroprotective, hepatoprotective, antidiabetic, and anthelmintics effects of different parts of CL received little attention. While there is still dearth of information on use of different parts of CL on cancer investigations and use. This scientific review on different parts of CL had highlighted knowledge gap that still exists on different parts of CL

Omic approaches to a better understanding of mastitis in dairy cows

Mastitis, which is caused by infection of the mammary gland is the most important disease problem facing dairy farmers. While the disease has been studied for decades in order to determine better diagnosis and treatment, it is only recently that the full panoply of advanced biotechnological methodologies has been applied that are needed to bring a systems biology approach to investigations. Molecular investigations using analyte-specific immunoassays, such as for the acute phase proteins haptoglobin and mammary-associated serum amyloid A3 in milk have introduced possibilities for monitoring the host inflammatory response. The omics revolution in biology, with genomics being harnessed especially for identification of the causative pathogens of mastitis, has enhanced dissection of the mammary microbiome. The application of proteomics, peptidomics and metabolomics to the diagnosis and pathophysiology of mastitis, in contrast, is in its infancy though the potential of these advanced tools of biological research is clear as they are applied in a systems biology analysis of this major health problem of dairy cows

Identifikacija promjena u ekspresiji proteina u mlijeku provedena razlikovnom elektroforezom u gelu kod eksperimentalno izazvanog goveđeg mastitisa

In order to identify the extent of protein changes in milk during mastitis as a guide to detecting markers for prompt management of the disease, milk from cows in which clinical mastitis was experimentally induced were subjected to difference gel electrophoresis (DiGE) analysis. Pooled samples from 6 udders (from 6 cows) were analysed at selected time points: 0, 81 and 312 hours post-challenge with Streptococcus uberis mastitis. These corresponded to samples from the pre-infection, peak and resolution phases of the mastitis challenge. After the preliminary sample preparation, concentration and pooling steps, samples were labelled with CyDyes (Cydye 2, 3 and 5), after which isoelectric focusing and gel electrophoresis were carried out respectively. DiGE gels were subsequently scanned and ImageQuant, ImageJ and DeCyder™ 2D (version 7.0) softwares were used to crop, obtain jpeg images and carry out 2-D differential analysis and processing of the images, respectively. Biological variation analysis (BVA) software (GE Healthcare Life Sciences, Buckinghamshire, UK) was also used to analyse the gels and create gel to gel matching of spots (qualitatively and quantitatively) within the three gels produced. Overall, a total of 521 protein spots were identified as significantly differentially expressed (qualitatively or quantitatively) in mastitis milk during the course of the intramammary infection. This demonstrates the large repertoire of protein biomarker candidates available, revealed through this technique. Further studies are required to elucidate the merits and demerits of these changing proteins in order to identify the one most suitable for clinical application in mastitis diagnosis.Cilj je bio ustanoviti opseg promjena u sastavu proteina mlijeka za vrijeme mastitisa i na taj način dati doprinos smjernicama za otkrivanje markera kojima bi se ubrzalo liječenje ove bolesti. Mlijeko krava u kojih je eksperimentalno izazvan klinički mastitis podvrgnuto je razlikovnoj elektroforezi u gelu (DiGE). Analizirani su objedinjeni uzorci iz 6 vimena (od 6 krava) u odabranim vremenskim točkama 0, 81 i 312 sati nakon infekcije bakterijom Streptococcus uberis. Navedene vremenske točke odgovaraju trima fazama mastitisa u kojima su uzeti uzorci: u predinfektivnoj fazi, u fazi u kojoj je infekcija dosegnula vrhunac i u fazi povlačenja mastitisa. Nakon preliminarne pripreme odnosno više koraka koncentracije i objedinjavanja, uzorci su obojeni pomoću CyDyes (Cydye 2, 3 i 5) te je provedeno izoelektrično fokusiranje i elektroforeza u gelu. DiGE gelovi zatim su skenirani te su primjenom softvera ImageQuant, ImageJ i DeCyder™ 2D (verzija 7.0) snimke obrezane, dobivene su jpeg slike i provedena je 2D diferencijalna analiza i obrada snimki. Upotrijebljen je i softver za analizu bioloških varijacija (BVA) (GE Healthcare life sciences, Buckinghamshire, UK) kako bi se gelovi analizirali i formiralo podudaranje točaka (kvalitativno i kvantitativno) unutar tri proizvedena gela. Utvrđeno je da je ukupno 521 mjesto proteina znakovito različito izraženo (kvalitativno i kvantitativno) u mlijeku krava s mastitisom za vrijeme intramamarne infekcije. Navedeno upućuje da je primjenjena tehnika ponudila veliki broj kandidatnih proteina koji mogu poslužiti kao biomarkeri mastitisa. Potrebna su daljnja istraživanja kako bi se razjasnile prednosti i nedostaci metode otkrivanja promjena u proteinima sa svrhom pronalaska najprikladnije kliničke primjene u dijagnostici mastitisa

Erythrogram, leukogram, and acute phase protein reference intervals for healthy newborn Murrah buffalo calves (Bubalus bubalis) within the first month of life

Establishing of reference intervals (RI) for hematologic variables and blood serum acute phase proteins (APP) of healthy newborn buffaloes is an important tool for monitoring alterations during infection and inflammation. Considering the scarcity of published data on newborns, the aim of the study was to establish RI for hematologic variables and APP from healthy newborn buffaloes. Blood samples from 28 healthy Murrah buffalo calves, 10–30 days old, were selected to determine RI. Fourteen hematologic and four blood APP variables were analyzed. Before collection of blood samples, calves were subjected to physical examination (rectal temperature, degree of dehydration, and fecal consistency) and only calves that were considered healthy were included in the study. The Anderson-Darling test was used to assess normal distribution of values. The Dixon test and Tukey test were used to identify outliers. RI and 90% CI were determined using standard/robust methods and Box-Cox transformation. RI for variables analyzed were the following: (1) hematologic variables: RBC 7.5–12.9 × 106/μL, HGB 10.6–19.0 g/dL, packed cell volume 33.1–54.8%, mean corpuscular volume 36.2–50.6 fL, mean corpuscular hemoglobin 12.1–17.3 pg, mean corpuscular hemoglobin concentration 28.1–42.9 g/dL, platelets 361–1081 × 103/μL, WBC 6.56–18.2 × 103/μL, lymphocytes 4.15–12.8 × 103/μL, segmented neutrophils 0.950–10.6 × 103/μL, band neutrophils 0–0.160 × 103/μL, monocytes 0–0.754 × 103/μL, eosinophils 0–0.326 × 103/μL, and basophils 0–0.149 × 103/μL and (2) APP variables: fibrinogen 2.49-9.50 g/L, haptoglobin 0.02-0.56 g/L, serum amyloid A (SAA) 3.70-97.51 μg/mL, and C-reactive protein (CRP) 0.02-2.78  μg/mL. In conclusion, hematologic and acute phase protein RI have been documented and can be used as a physiologic database to help the interpretation of laboratory results of newborn buffaloes during infection and inflammation conditions

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

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 &#60;0.4–55 μg/ml with a median of 3.5 μg/ml; composite milk M-SAA3 ranged from &#60;0.6–50 μg/ml and had a median of 1.2 μg/ml, while CRP ranged from &#60;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 &#60;0.009) as well as parity and Hp (P &#60; 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 &#60;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

Effects of pre-operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care. We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care