Subclinical Mastitis Group Research Articles

Changes in the Microbiome in Yak Mastitis: Insights Based on Full-Length 16S rRNA Sequencing.

Mastitis is an inflammation of the mammary gland that can be caused by various factors, including biological, chemical, mechanical, or physical. Microbiological culture, DNA techniques, and high-throughput next-generation sequencing have been used to identify mastitis-causing pathogens in various animal species. However, little is known about microbiota and microbiome changes linked to yak milk mastitis. This study aimed to characterize the milk microbiota of healthy and mastitis-infected yaks using full-length 16S rRNA sequencing. The results showed that the bacterial microbiota comprises 7 phyla, 9 classes, 20 orders, 39 families, 59 genera, and 72 species. Proteobacteria and Firmicutes were the predominant microbial communities, with lower abundances of Bacteroidota, Actinobacteriota, Acidobacteriota, and other minor groupings also observed. Proteobacteria dominated the clinical and subclinical mastitis groups (95.36% and 89.32%, respectively), in contrast to the healthy group (60.17%). Conversely, Firmicutes were more common in the healthy group (39.7%) than in the subclinical and clinical mastitis groups (10.49% and 2.92%, respectively). The predominant organisms found in the healthy group were Leuconostoc mesenteroides, Lactococcus piscium, Carnobacterium maltaromaticum, and Lactococcus raffinolactis. Low abundances of Staphylococcus aureus species were found in both subclinical and clinical mastitis groups, with Moraxella osloensis and Psychrobacter cibarius dominating the subclinical mastitis group and Pseudomonas fluorescens dominating the clinical mastitis group. An alpha diversity study revealed that the healthy group had a higher microbial diversity than the clinical and subclinical mastitis groups. According to beta-diversity analysis, the principal coordinate analysis identified that mastitis-infected samples significantly differed from healthy ones. The milk microbiota of healthy yaks is more varied, and specific prominent taxa within various groups can act as marker microorganisms for mastitis risk. The genera Leuconostoc and Lactococcus are promising candidates for creating probiotics.

Subclinical mastitis in Jersey dairy cows and its effects on productivity and inflammatory markers

This study assessed the effect of subclinical mastitis on the productivity and inflammatory markers in Jersey dairy cows. Blood, milk, and milk yield data were collected from 59 Jersey dairy cows reared under a semi-extensive system. Milk samples were collected from individual collectors and evaluated for their somatic cell count (SCC), lactose (Lact), protein, fat, total and defatted dry extracts (DDE), casein, freezing point, and milk urea nitrogen (MUN) levels. After milking, blood was collected by puncturing the coccygeal arteriovenous complex. In the serum samples, the inflammatory biomarkers paraoxonase-1, albumin, and total plasma protein levels were analyzed using colorimetric methods. Samples of the entire diet provided and pastures were submitted for bromatological analysis. Additionally, the body condition score, number of lactations, milk yield, days of lactation, calving interval, number of inseminations until conception, calving-conception interval, and days of pregnancy were analyzed. The cows were categorized based on their SCC into a subclinical mastitis group (SubG), with SCC levels greater than or equal to 200,000 cells/mL, and a control group (CG), with SCC levels below 200,000 cells/mL. Blood metabolic variables, milk quality, and milk production data were analyzed using multivariate regression, analysis of variance, and logistic regression using the SAS® program. The multivariate regression analysis revealed that several markers, such as Lact, total protein, and casein, influenced milk composition, predicting over 97% of the data. The SubG showed lower concentrations of Lact (4.37 vs. 4.47%; P = 0.0002), MUN (20.55 vs. 23.85 mg/dL; P = 0.02), and DDE (9.24 vs. 9.50%; P = 0.02) compared with the CG; moreover, animals in the SubG had a higher number of lactations compared with those in the CG (4.30 vs. 2.69; P = 0.0039). Cows with a Lact content lower than 4.265% (quartile 25%), compared with those with Lact content greater than 4.565% (quartile 100%), were 52 times more likely to have subclinical mastitis. Jersey dairy cows with subclinical mastitis have lower Lact, urea, and defatted dry extract levels in their milk as well as a higher number of lactations. Therefore, Lact levels in milk serve as a good diagnostic marker of subclinical mastitis in Jersey cows. Subclinical mastitis in Jersey dairy cows did not decrease milk yield nor effect reproductive performance.

Effect of subclinical mastitis on milk taurine concentration in dairy cows

The objective of this study was to evaluate whether taurine has any role in bovine subclinical mastitis, and the relationship between milk taurine concentration and somatic cell count. Forty milk samples from dairy cows aged 3 to 6 during lactation were used for this research. The California Mastitis Test (CMT) and somatic cell counts (SCC) were determined in the diagnosis of subclinical mastitis. The subclinical mastitis group consisted of 20 milk specimens positive for CMT and SCC>200.000/ml of milk. The control group consisted of 20 milk specimens negative for CMT and SCC<200.000/ml of milk. A bovine-specific taurine enzyme-linked immunosorbent assay kit was used to determine the taurine concentration in the milk. The milk serum taurine concentrations were determined as 232.1±89.8 pg/ml in the control group and 158.0±55.6 pg/ml in the subclinical mastitis group. These results determined that the taurine concentration in milk with subclinical mastitis decreased approximately 1.5 times compared to healthy milk (P<0.05). A significant negative correlation was determined between the taurine concentration in cow’s milk with subclinical mastitis and the number of somatic cells (r= -0.933; P<0.001). From the findings of this study, it was concluded that both somatic cell counts and the measurement of taurine in cow’s milk could be used to monitor mammary health.

Case-Control Study: Endogenous Procalcitonin and Protein Carbonylated Content as a Potential Biomarker of Subclinical Mastitis in Dairy Cows.

Procalcitonin (PCT) and protein carbonylated content (PCC) are promising biomarkers for bacterial infection and inflammation in veterinary medicine. This study examined plasma PCT and PCC levels in healthy cows (H) and cows with subclinical mastitis (SCM). A total of 130 cows (65 H and 65 SCM) were included in this study. Blood samples were collected, and plasma was frozen at -80 °C. PCT levels were determined using a bovine procalcitonin ELISA kit, while PCC was measured following the methodology of Levine et al. Statistical analysis revealed a significant difference in PCT levels between H (75.4 pg/mL) and SCM (107.3 pg/mL) cows (p < 0.001) and significantly lower concentrations of PCC in the SCM group (H: 0.102 nmol/mL/mg, SCM: 0.046 nmol/mL/mg; p < 0.001). The PCT cut-off value for distinguishing healthy and subclinical mastitis animals was >89.8 pg/mL (AUC 0.695), with a sensitivity of 66.2% and specificity of 69.2%. PCT showed potential value as a diagnostic tool to help in decision making for subclinical mastitis cases, while PCC requires further studies to investigate the trend of this biomarker during localized pathology.

Investigation of milk microbiota of healthy and mastitic Sahiwal cattle

BackgroundSahiwal cattle is an indigenous cattle breed of Pakistan and mastitis is one of the major problems faced by Sahiwal cattle which hinders its production potential. The study was designed to investigate the milk microbiota of healthy and mastitic Sahiwal cattle as part of a multistep project to develop probiotics for the mitigation and control of mastitis. Milk samples of Sahiwal cattle (healthy clinical mastitis and subclinical mastitis) reared under similar husbandry and management practices were processed for 16S rRNA gene base metagenomics analysis.ResultsResults revealed that Proteobacteria were dominant in the healthy group and subclinical mastitis group (56.48% and 48.77%, respectively) as compared to the clinical mastitis group (2.68%). In contrast, Firmicutes were abundant in the clinical mastitis group (64%) as compared to the healthy and subclinical mastitis groups (15.87% and 38.98%, respectively). Dominant species assigned in the healthy group were Ignavibacterium album, Novosphingobium capsulatum, Akkermansia muciniphila and Lactobacillus fermentum.The clinical mastitis group was dominated by Streptococcus dysgalactiae and Corynebacterium bovis, while subclinical mastitis group included Lactobacillus fermentum and uncultured acidobacteriales and Akkermansia muciniphila as dominant species. Alpha diversity indices showed higher microbial diversity in the healthy group compared to the clinical and sub-clinical mastitis groups.ConclusionIt is concluded that the milk microbiota of healthy sahiwal cattle has higher diversity and dominant taxa in the different groups may be used as signature microbes for mastitis susceptibility. Akkermansia muciniphila is one of candidate specie that was identified and may be used for development of probiotics.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.

Biochemical and Computational Assessment of Acute Phase Proteins in Dairy Cows Affected with Subclinical Mastitis.

Subclinical mastitis (SCM) is a predominant form of mastitis wherein major visible signs of disease are absent. The present study aimed to determine acute phase proteins (APPs) like ferritin, C-reactive protein (CRP), and microalbumin (Malb) in 135 composite milk and serum samples of healthy (n = 25) and SCM (n = 110) cows. As bovine mastitis is an inflammatory disease, the present study also aimed at finding novel anti-inflammatory compounds from natural sources by repurposing approach using computational studies. The findings of the present study revealed substantial elevation (p < 0.001) in milk SCC and an increase in ferritin, CRP, and Malb (p < 0.001) in milk and sera of the SCM group as compared to healthy animals. Receiver operating characteristics of milk SCC, milk, and serum APPs unraveled statistically substantial alteration (p < 0.001). Further, SCC was correlated with milk APPs ferritin (r = 0.26 **, p < 0.002), CRP (r = 0.19 *, p < 0.02), and Malb (r = 0.21 *, p < 0.01). Additionally, milk SCC was correlated with serum ferritin (r = 0.28 **, p < 0.001), CRP (r = 0.16, p > 0.05), and Malb (r = 0.16, p > 0.05). The findings of molecular docking revealed that Chaetoglobosin U was the most effective molecule that showed the highest binding affinity (kcal/mol) of -10.1 and -8.5 against ferritin and albumin. The present study concluded that the estimation of cow-side tests, SCC, and APPs in milk/serum is suitable to detect SCM and screening herd community. Furthermore, Chaetoglobosin U could be developed as a promising anti-inflammatory inhibitor; however, further studies are required to validate these findings.

Assessment of milk and blood enzymes in Holstein cows with subclinical mastitis

The objective of this study was to determine the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the milk and blood of lactating Holstein cows and investigate their association with subclinical mastitis (SCM). This study also aims to evaluate the impact of SCM and its severity based on California mastitis test (CMT) scores on the above mentioned enzymes, as well as to test the efficiency of their use as an early predictive diagnostic tool. Thirty multiparous, lactating dairy Holstein cows were examined using CMT and according to the obtained results classified into four groups; control group 1 (SCM-free = 10, score = 0), group 2 (weak positive = 8, score +1), group 3 (positive = 7, score +2) and group 4 (strong positive = 5, score +3). The milk LDH, ALP, and GGT activities were significantly higher in groups 3 and 4 compared to group 2 and the control group. LDH activity was significantly higher in group 2 compared to the control group. The mean blood LDH and ALP activities in SCM groups were significantly higher than in the control group. ALP blood activity was significantly higher in group 4 compared to group 2. The blood GGT activity was significantly higher in group 4 compared to other groups. There was a significant positive association between milk LDH, ALP, and GGT enzymes as well as the blood enzymes (ALP and GGT) in SCM cows. The results showed that subclinical mastitis causes considerable changes in the activities of enzymes in milk and blood of infected cows.

Comparative Proteomic Analysis of Milk-Derived Extracellular Vesicles from Dairy Cows with Clinical and Subclinical Mastitis.

Extracellular vesicles (EVs) are membranous vesicles found in biological fluids with essential functions. However, milk-derived EV proteins from clinical mastitis (CM) and subclinical mastitis (SM) cows have yet to be studied in detail. In this study, milk-derived EVs of CM, SM, and Healthy cows were extracted using a combination of acetic acid/ultracentrifugation and density gradient ultracentrifugation and analyzed using a shotgun proteomic by data-independent acquisition mode. A total of 1253 milk exosome proteins were identified and quantified. Differently enriched (DE) proteins were identified as given a Benjamini−Hochberg adjusted p < 0.05 and a fold change of at least 2. There were 53 and 1 DE proteins in milk-derived EVs from CM and SM cows compared with healthy cows. Protein S100-A9, Protein S100-A8, Chitinase-3-like protein 1, Haptoglobin, Integrin beta-2, and Chloride intracellular channel protein 1 were more abundant in the CM group (adjusted p < 0.05). Still, their enrichment in the SM group was not significant as in the Healthy group. The enrichment of DE proteins between CM and Healthy group was consistent with elevated GO (Gene Ontology) processes—defense response, defense response to Gram-positive bacterium, granulocyte chemotaxis also contributed to Reactome pathways—neutrophil degranulation, innate immune system, and antimicrobial peptides in the CM group. These results provide essential information on mastitis-associated proteins in milk-derived EVs and indicate the biological functions of milk-derived EVs proteins require further elucidation.

Genome-Wide DNA Methylation and Its Effect on Gene Expression During Subclinical Mastitis in Water Buffalo.

Subclinical mastitis (SCM) in buffalo is one of the most challenging paradoxes for the dairy sector with very significant milk production losses and poses an imminent danger to milch animal’s milk-producing ability. We present here the genome-wide methylation specific to SCM in water buffalo and its consequential effect on the gene expression landscape for the first time. Whole-genome DNA methylation profiles from peripheral blood lymphocytes and gene expression profiles from milk somatic cells of healthy and SCM cases were catalogued from the MeDIP-Seq and RNA-Seq data. The average methylation in healthy buffaloes was found to be higher than that in the SCM-infected buffaloes. DNA methylation was abundant in the intergenic region followed by the intronic region in both healthy control and SCM groups. A total of 3,950 differentially methylated regions (DMRs) were identified and annotated to 370 differentially methylated genes (DMGs), most of which were enriched in the promoter region. Several important pathways were activated due to hypomethylation and belonged to the Staphylococcus aureus infection, Th17 cell differentiation, and antigen processing and presentation pathways along with others of defense responses. DNA methylome was compared with transcriptome to understand the regulatory role of DNA methylation on gene expression specific to SCM in buffaloes. A total of 4,778 significant differentially expressed genes (DEGs) were extracted in response to SCM, out of which 67 DMGs were also found to be differentially expressed, suggesting that during SCM, DNA methylation could be one of the epigenetic regulatory mechanisms of gene expression. Genes like CSF2RB, LOC102408349, C3 and PZP like, and CPAMD8 were found to be downregulated in our study, which are known to be involved in the immune response to SCM. Association of DNA methylation with transposable elements, miRNAs, and lncRNAs was also studied. The present study reports a buffalo SCM web resource (BSCM2TDb) available at http://webtom.cabgrid.res.in/BSCM2TDb that catalogues all the mastitis-related information of the analyses results of this study in a single place. This will be of immense use to buffalo researchers to understand the host–pathogen interaction involving SCM, which is required in endeavors of mastitis control and management.

Blood Metabolomic Phenotyping of Dry Cows Could Predict the High Milk Somatic Cells in Early Lactation—Preliminary Results

Subclinical mastitis (SCM) is a very common disease of dairy cows. Currently, somatic cell count (SCC) is used for SCM diagnoses. There are no prognostic tests to detect which cows may develop SCM during the dry-off period. Therefore, the objectives of this study were to identify metabolic alterations in the serum of pre-SCM cows during the dry-off period, at −8 and −4 weeks before calving, through a targeted mass spectrometry (MS) assay. Fifteen cows, free of any disease, and 10 cows affected only by SCM postpartum served as controls (CON) and the SCM group, respectively. Results showed 59 and 47 metabolites that differentiated (p ≤ 0.05) CON and pre-SCM cows at –8 and −4 weeks prior to the expected date of parturition, respectively. Regression analysis indicated that a panel of four serum metabolites (AUC = 0.92, p &lt; 0.001) at −8 weeks and another four metabolites (AUC = 0.92, p &lt; 0.01) at −4 weeks prior to parturition might serve as predictive biomarkers for SCM. Early identification of susceptible cows can enable development of better preventive measurements ahead of disease occurrence.

Changes in social and feeding behaviors, activity, and salivary serum amyloid A in cows with subclinical mastitis

The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors, such as decreased activity, feeding and drinking (here labeled "core maintenance" behaviors), and less well-studied social, grooming, and exploratory behaviors (here labeled "luxury" behaviors). Luxury behaviors are biologically predicted to change at lower levels of mastitis infection and are, therefore, particularly relevant to detecting subclinical mastitis. Salivary serum amyloid A is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for 17 matched pairs of commercial barn-housed Holstein-Friesian cows. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC; SCM: SCC >200 × 1,000 cells/mL; CTRL: SCC <100 × 1,000 cells/mL). SCM cows were selected for study ad hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 d: SCC (d 1), behavior (d 2), salivary SAA (d 3). All behaviors performed by the focal cows over a single 24-h period were coded retrospectively from video footage, and differences between the SCM and CTRL groups were investigated using the main data set and a subset of data corresponding to the hour immediately following morning food delivery. Saliva was collected using cotton swabs and analyzed for SAA using commercially available ELISA kits. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows displayed reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic noncontact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. The SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with 2 conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription–polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

Prevalence of Subclinical Mastitis and its Effects on Reproductive Performance in Dairy Cows during the Postpartum Period in Gasabo District, Rwanda

Mastitis is one of the most important diseases affecting production and reproductive efficiency of dairy cows. The objective of this study was to estimate the prevalence of subclinical mastitis (SCMA) and its effects on reproductive performance in postpartum dairy cows. In a cross-sectional survey, sample cows (n = 200) within their 10-40 days in milk from smallholder farms (n = 159) were selected and tested for SCMA using California Mastitis Test. They were classified into two groups: cows with SCMA (SCMA group, n = 101) and cows without SCMA (WSCMA group, n = 99). Reproductive performance including calving-to-first-oestrus interval (CFOI), calving-to-first service interval (CFSI), calving-to-conception interval (CCI), cows that remained non-pregnant, postpartum anoestrus, and total number of services per conception were recorded for 150 days in milk. The overall prevalence of SCMA recorded was 50.5%. The mean number of days for CFOI was significantly longer (p

Use of the Rumination Profile Through Collar Sensors for Mastitis Diagnosis in Dairy Cows

Background: Mastitis is an inflammatory disease of the mammary gland, mostly associated with bacterial infections. It is responsible for great economic losses due to decreased milk yield, discarded milk, milk composition alterations and treatment costs, besides it impairs the animal health and welfare. The rumination time is an important behavioral marker and its assessment can be used as an early diagnosis tool, which can improve cure rate. Therefore, the aim of the present study was to evaluate the sensitivity of behavior monitoring system collars in the diagnosis of mastitis and the average rumination time (RT) of Holstein cows during the healthy period and affected by the disease.Materials, Methods &amp; Results: The study was conducted on a commercial property located in the municipality of Rio Grande, Rio Grande do Sul, Brazil. The RT data from 39 multiparous Holstein cows with an average milk yield of 38.4 L/day was collected. RT monitoring was performed using C-Tech1 collars combined with CowMed® software, which assess behavior data from the animals and emits warning signals when it finds abnormalities in any parameter. In order to verify whether the animals were determined correlated with diseases, the sensitivity of the data was evaluated, when the system had given the alert to animals considered ill, they underwent to a further clinical evaluation performed by a veterinarian to confirm the diagnosis. From the diagnosis, the cows were divided into subclinical mastitis (SM) and clinical mastitis (CM) groups. SM was detected by the Californian Mastitis Test (CMT) and cows that were graded 1 (++), 2 (++) or 3 (+++) without the presence of any other clinical sign were assigned to the SM group. CM was assessed by observation of abnormalities in milk such as changes in color and consistency, as well as the presence of lumps, clots or blood; and clinical examination of the udder was performed for detection of hot, hard, swollen or painful quarters. Thereafter, variations in the mean RT between the healthy (15 days) and sick periods (days when there was an alert) were evaluated. For the identification of the etiological agents involved in the cases of CM, microbiological cultures were performed on Accumast® plates with milk samples, which were incubated at a temperature of 37°C and the diagnosis of the pathogens were performed after16 h of incubation. During the study, 57 cases of mastitis were observed, 42 were SM and 15 were CM. The sensitivity of the system, which is the ability to detect positive cases of the disease, was 73.8% for SM and 73.3% for MC. The RT of the animals were compared individually during the healthy period with the sick period and it was observed that SM reduced the RT by 5.33% whereas MC reduced the RT by 14.9%.Discussion: The maximum RT values were lower during the disease period in relation to the period in which the animals were healthy, for both SM and CM, which is due to the fact that the disease is responsible for causing inappetence in animals, among other clinical signs, therefore, reducing feed consumption. The lowest variation in RT, between the healthy and sick period, was observed in cases of SM, which was already expected, since the clinical form tends to cause more discomfort to the animals. As for the main etiological agents involved in the clinical condition, Streptococcus agalactiae and S. uberis were detected. In view of the above, the evaluation of the mean RT of multiparous dairy cows was efficient in the predictive diagnosis of SM and CM up to 2 days before the onset of the disease. In addition, the variability of this result demonstrated that animals with subclinical cases presented less fluctuation in RT.

Nitric Oxide Molecule as A Potential Target of Alternative Strategies for the Diagnosis of Subclinical Mastitis in Cows

The mastitis is an important disease affecting daily milk yield and quality in lactating cows, it negatively affects both animal welfare and economic income. The aim of this study is to investigate the effect of subclinical mastitis on nitric oxide (NO), total antioxidant capacity (TAC), total oxidant capacity (TOC), albumin, total protein, globulin, glucose and gamma glutamyl transpeptidase activity (GGT) levels in plasma. The research was carried out in Brown Swiss and Holstein cows. The mammary lobes of the animals were scanned with the California Mastitis Test (CMT) and those with subclinical mastitis were identified. Samples with CMT (-) were classified as healthy (n = 10) and those with CMT (+) were classified as subclinical mastitis group (n = 10). The study presented is a basic feature and results clearly showed that significant amounts of NO was released in plasma during subclinical mastitis. There was no statistically significant difference between cows with or without subclinical mastitis for plasma TAC, TOC, albumin, total protein, globulin, glucose, albumin/globulin levels and GGT activity. Consequently, nitric oxide is an important mediator in subclinical mastitis as a potential target of therapeutic strategies and can be evaluated in alternative diagnosis.

Subclinical Mastitis in a European Multicenter Cohort: Prevalence, Impact on Human Milk (HM) Composition, and Association with Infant HM Intake and Growth.

Background: Subclinical mastitis (SCM) is an inflammatory condition of the mammary gland. We examined the effects of SCM on human milk (HM) composition, infant growth, and HM intake in a mother–infant cohort from seven European countries. Methods: HM samples were obtained from 305 mothers at 2, 17, 30, 60, 90, and 120 days postpartum. SCM status was assessed using HM Sodium (Na): Potassium (K) ratio >0.6. Levels of different macro- and micronutrients were analyzed in HM. Results: SCM prevalence in the first month of lactation was 35.4%. Mean gestational age at delivery was lower and birth by C-section higher in SCM mothers (p ≤ 0.001). HM concentrations of lactose, DHA, linolenic acid, calcium, and phosphorous (p < 0.05 for all) was lower, while total protein, alpha-lactalbumin, lactoferrin, albumin, arachidonic acid to DHA ratio, n-6 to n-3 ratio and minerals (iron, selenium, manganese, zinc, and copper) were higher (p < 0.001 for all) in mothers with SCM. There were no differences in infant growth and HM intake between non-SCM and SCM groups. Conclusion: We document, for the first time, in a large European standardized and longitudinal study, a high prevalence of SCM in early lactation and demonstrate that SCM is associated with significant changes in the macro- and micronutrient composition of HM. Future studies exploring the relation of SCM with breastfeeding behaviors and developmental outcomes are warranted.

Prediction of quarter level subclinical mastitis by combining in-line and on-animal sensor data

We investigated the potential for automatic detection of subclinical mastitis (SCM) in pasture-based automatic milking systems. The objective of the study was to determine the ability of electrical conductivity (EC), together with relative changes in daily activity (activity) and daily rumination (rumination) recorded using heat and rumination–long-distance tags, to predict quarter-level SCM. Activity (arbitrary unit/day) and rumination (min/day) data were determined across 21 days using heat and rumination–long-distance tags for 170 cows. Cows were allocated into the following three groups: SCM (n = 32, EC ≥ 7.5 millisiemens/cm (mS/cm) in one or more quarters and a positive bacteriological culture in the corresponding quarter(s)); true-negative (TN, n = 9, EC ≥ 7.5 mS/cm and a negative culture in all four quarters); and apparently healthy (n = 129, no culture test and EC &amp;lt; 7.5 mS/cm). Group mean differences in activity and rumination were compared using Welch’s t-tests. Logistic mixed models were used to predict SCM by EC, activity and rumination changes before mastitis detection, including parity information between SCM and TN groups. Cow- and quarter-specific information were included as random effects, followed by model assessment by producing receiver operating-characteristic curve and area under the curve (AUC) value. In total, 287 quarters were used in the prediction model, including 143 quarters with a positive culture (Gram-positive; n = 131, Gram-negative; n = 6, mixed; n = 6) and 144 quarters with a negative culture. On average, SCM group had 4.65% greater (P &amp;lt; 0.01) activity and 9.89% greater (P &amp;lt; 0.001) rumination than did the TN group and 11.70% greater (P &amp;lt; 0.001) activity than did the apparently healthy group. A combined model with terms for EC, activity changes, rumination changes prior to detect SCM and parity had a better SCM prediction (AUC = 0.92) ability than did any of them separately (all AUC &amp;lt; 0.8). Hence, we conclude that EC in combination with activity and rumination information can improve the accuracy of prediction of quarter-level SCM.

Paraoxonase 1 in bovine milk and blood as marker of subclinical mastitis caused by Staphylococcus aureus

The aim of this study was to determine serum lipid values and parameters of oxidative stress in blood and milk of cows with subclinical mastitis (SCM) caused by Staphylococcus aureus and to establish association between these parameters. The study was performed on total of 104 cows assigned into control group of healthy animals (n = 12) and two groups of cows with SCM, either SCM1 group (n = 37) with ˂ 1000 CFU/mL of S. aureus or SCM2 group (n = 55) with ≥1000 CFU/mL of causative agent in milk. Significantly lower serum concentrations of high density lipoprotein-cholesterol (HDLC) in SCM2 group and higher low density lipoprotein-cholesterol (LDL-C) in both SCM groups were recorded. Significantly lower paraoxonase-1 (PON1) activity and higher lipid hydroperoxides (LOOH) concentration in blood and milk were recorded in both groups of SCM cows vs control. In blood serum of cows from SCM2 group significantly higher concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA), but significantly lower total antioxidative capacity (TAC) were obtained. In milk serum of SCM cows were detected significantly higher concentrations of H2O2 and MDA, but significantly lower TAC vs control. There was a significant positive correlation between PON1 in serum with HDLC, but negative correlation with LDL-C and LOOH. In milk, PON1 negatively correlated with LOOH and somatic cell counts (SCC). Strong positive correlation was obtained between PON1 in blood and milk. Oxidative stress and inflammatory reaction induced by SCM significantly lowered PON1 activity in blood and milk of affected cows.

Paraoxonase-1 activity and lipid profile in dairy cows with subclinical and clinical mastitis

ABSTRACT The aim of this study was to investigate serum paraoxonase-1 (PON1) activity and lipid status in cows with subclinical and clinical mastitis in order to evaluate systemic inflammatory and oxidative stress responses. The study was conducted on a total of 90 Holstein–Friesian dairy cows kept in farms in eastern Croatia. Cows were assigned into three groups: the cows suffering from clinical mastitis (CLM), the cows with subclinical mastitis (SCM) and control (CTL) group. In collected sera, PON1, lipid status and calcium concentration were measured. Total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C) and calcium concentrations were significantly lower in the CLM group of cows compared to the SCM and CTL groups (P < 0.05). There were no significant differences in the lipid status and calcium level between the CTL and SCM groups. PON1 activity was significantly lower in both the SCM and CLM groups compared with CTL indicating that PON1 could be considered as a potential biomarker for the diagnosis of subclinical form of the disease.