Clinical Mastitis Research Articles

Unravelling the complexity of bovine milk microbiome: insights into mastitis through enterotyping using full-length 16S-metabarcoding

BackgroundMastitis, inflammation of the mammary gland, is a major disease of dairy cattle and the main cause for antimicrobial use. Although mainly caused by bacterial infections, the aetiological agent often remains unidentified by conventional microbiological culture methods. The aim of this study was to test whether shifts in the bovine mammary gland microbiota can result in initiation or progression of mastitis.MethodsOxford-Nanopore long-read sequencing was used to generate full-length 16S rRNA gene reads (16S-metabarcoding) to characterise the microbial population of milk from healthy and diseased udder of cows classified into five groups based on their mastitis history and parity.ResultsSamples were classified into six enterotypes, each characterised by a marker genus and several differentially-abundant genera. Two enterotypes were exclusively composed of clinical mastitis samples and displayed a marked dysbiosis, with a single pathogenic genus predominating and displacing the endogenous bacterial population. Other mastitis samples (all subclinical and half of the clinical) clustered with those from healthy animals into three enterotypes, probably reflecting intermediate states between health and disease. After an episode of clinical mastitis, clinical recovery and microbiome reconstitution do not always occur in parallel, indicating that the clinical definition of the udder health status does not consistently reflect the microbial profile.ConclusionsThese results show that mastitis is a dynamic process in which the udder microbiota constantly changes, highlighting the complexity of defining a unique microbiota profile indicative of mastitis.

Whole genome sequence analysis of multi-drug resistant and biofilm-forming Staphylococcus haemolyticus isolated from bovine milk

BackgroundMilk is an excellent growth medium for microorganisms due to its nutritive composition. Microorganisms have been implicated in bovine mastitis (BM) in dairy cows as well as causing infections in animals and humans. Despite extensive endeavours to manage BM, this condition continues to persist as the most prevalent and economically burdensome problem affecting dairy cattle on a global scale. Non-aureus staphylococci (NAS) species such as Staphylococcus haemolyticus, S. epidermidis, and S. xylosus are currently the predominant microbiological agents identified as the main cause of subclinical udder infections and are also considered opportunistic pathogens in cases of clinical mastitis in dairy cows. Therefore, it is crucial to elucidate the genetic profile of these species. The primary objective of this study was to characterise three phenotypically determined multidrug-resistant NAS environmental strains (NWU MKU1, NWU MKU2, and NWU MKS3) obtained from dairy cows milk via whole-genome sequencing.ResultsThe results confirmed that the three isolates were S. haemolyticus with genome sizes of 2.44, 2.56, and 2.56 Mb and a G + C content of 32.8%. The genomes contained an array of antibiotic resistance genes that may potentially confer resistance to a range of antibiotic classes, such as macrolides, fluoroquinolones, aminoglycosides, cephalosporins, tetracyclines, peptides, and phenicol. Furthermore, all the genomes carried virulence genes, which are responsible for several functions, such as adhesion, enzyme and toxin production. The genomes of these organisms contained signatures encoding mobile genetic elements such as prophages and insertion sequences.ConclusionThese findings indicate there is a need for diligent monitoring with improved management practices and quality control strategies on farms to safeguard milk production systems and human health.

Whole-genome sequencing of Streptococcus uberis isolated from cows with mastitis in Thuringia.

Introduction. Streptococcus uberis is a common cause of mastitis in cattle, leading to significant economic losses. The widespread use of antimicrobials has contributed to the emergence of resistance, which poses a severe challenge in controlling S. uberis infection.Aim. The objective of this study was to gain insights into the antimicrobial resistance (AMR) and epidemiological typing of S. uberis isolated from milk collected from bovine mastitis on dairy farms in Thuringia.Methodology. In this study, 84 S. uberis isolates were obtained from cattle with clinical mastitis in Thuringia, their phenotypic and genotypic AMR were analyzed and their phylogenetic relationship was explored using whole-genome sequencing.Results. Genetically heterogeneous strains were found on the farms, but clusters of highly similar strains also circulated within the same farms. All isolates were sensitive to ampicillin, penicillin, ceftiofur, and vancomycin. However, 42.9%, 42.9%, 22.6%, 19.0%, and 13.0% were resistant to tetracycline, doxycycline, clindamycin, pirlimycin, and erythromycin, respectively. Thirty-nine strains were phenotypically resistant to two or more tested antibiotics. We identified a plasmid associated with macrolide and lincosamide resistance in 12% of the strains.Conclusion. The emergence of S. uberis strains resistant to multiple antibiotics highlights the importance of S. uberis surveillance and the prudent use of antimicrobials.

Draft genome sequencing of multidrug-resistant Pseudomonas asiatica strains isolated from dairy cows with clinical mastitis and their farm environment.

We report, for the first time, the draft genomes of four multidrug-resistant Pseudomonas asiatica strains isolated from milk (2M1), feces (2F1 and 2F2), and farm soil (2S1) samples of dairy cows with clinical mastitis. The assembled genomes of these strains were 5.7 Mbp, 62.8% G + C content, and 50× genome coverage.

Draft genome sequencing of multidrug-resistant Pseudomonas putida strains, isolated from dairy cows with clinical mastitis and their farm environment.

We sequenced the genomes of three multidrug-resistant Pseudomonas putida strains namely 11CM-M1, 11CM-F1, and 11CM-S1, isolated from milk, feces, and farm soil samples collected from dairy cows with clinical mastitis. The assembled draft genomes of these P. putida strains were approximately 5.6 Mbp, with a GC content of 62.4%.

Prevalence and Bacterial Isolation Causing Clinical and Subclinical Mastitis in Egyptian Dairy Cow in Kafer El-Sheikh, Egypt

Prevalence and Bacterial Isolation Causing Clinical and Subclinical Mastitis in Egyptian Dairy Cow in Kafer El-Sheikh, Egypt

The causes of Clinical and Subclinical Pathogenic Mastitis and anti-bacterial Resistance in Milk of Dairy Cows in and around Shahat, Libya

: من إجمالي 200 عينة حليب جمعت في الفترة ما بين يناير 2018 إلى مارس 2020 تم عزل 126 عينة مصابة بالتهاب بكتيري بنسبة (63٪. (مقسمة كالتالي: كأكبر عامل مسبب لالتهاب الضرع السريري وهي: Escherichia coli 46 (36.5%), Streptococcus pyogens 31 (24. 6%), and positive staphylococcus aureus 19 (15 %). بينما Streptococcus dysgalactiae(15.079. 21%) كانت المسبب الأكبر في التهاب الضرع الإكلينيكي. مع ذلك، وفي كلا النوعين من التهاب الضرع، تم أيضًا عزل 4 حالة إيجابية من عوامل بكتيرية أخرى والتي كانت بنسبة منخفضه، وبالتالي لا يمكن اعتبارها مهمة مثل Klebsiella spp أو غير معدية مثل Corynebacterium spp.وكانت مقاومة البكتريا للمضادات الميكروبية معتدلة في إجمالي العينات إلا penicillin وكذلك tetracycline في Streptococci وفي Staphylococci النسبة وإن كانت ليست عالية إلا أنها تهدد الصحة العامة ويرجع أهمية هذه المضادات الحيوية إنها تستخدم في علاج البشر بالإضافة إلى أنه يجب التنويه إلى أن المعلومات حول مشكلة التهاب الضرع في ليبيا ضعيفة.

Marker weighting improves single-step genomic prediction reliabilities of udder health traits in Nordic Red and Jersey dairy cattle populations

The standard single-step genomic prediction model assumes that all SNP markers explain an equal amount of genetic variance, which, however, may not be true. This is because SNPs are located in or near different genes with different functions. Therefore, it seems logical to consider SNP marker-specific weights when predicting genomic breeding values. We hypothesized that allowing differences in the amount of genetic variance explained by each SNP marker will improve prediction reliability and response to selection. To investigate this hypothesis, we first developed multi-trait standard single-step genomic models based on the current multi-trait random regression evaluation models for udder health traits of the Nordic Red (RDC) and Jersey (JER) dairy cattle populations. The models included 4 clinical mastitis (CM) traits, 3 test-day somatic cell score (SCS) traits, and the conformation traits fore udder attachment and udder depth. In the second step, we investigated the effect of applying different SNP marker weighting scenarios in the single-step genomic prediction models, for which a single-step SNP best linear unbiased prediction model was applied. We investigated the prediction reliability of the different models by forward prediction, where the last 4 years of the data were removed to estimate breeding values for validation candidates. In addition, genetic trends of the pedigree-based estimated breeding values (PEBV) and genomic enhanced breeding values (GEBV) were examined. The data sets for RDC and JER included 6.9 and 1.2 million animals of which 5.6 and 0.9 million cows had records, respectively. The number of genotyped animals was 125,789 and 64,777 for RDC and JER, respectively. Cows had repeated SCS observations but only single observations for all other traits and breeding values for all traits were modeled by one covariance function. This required modeling 12 eigenvalue breeding value coefficients for each cow and developing SNP marker weights for the principal components rather than for the biological traits. We investigated 3 SNP marker weighting scenarios: 1) a nonlinear method similar to BayesA, 2) using the classical formula 2pqû2 that accounts for allele heterozygosity, and 3) applying a mean SNP weight calculated by 2pqû2 for every 20 adjacent SNP markers. Bias, dispersion, and prediction reliability were calculated using PEBV or GEBV from the evaluation based on the full data set on those using the reduced data set. We found that the recent favorable genetic trend in CM and SCS has been accelerated since the introduction of genomic selection. The study also shows that a significant increase in prediction reliability, i.e., 0.74 vs. 0.48 for RDC and 0.72 vs. 0.41 for JER cows for CM, can be achieved with a standard single-step genomic prediction model compared with a pedigree-based prediction model. Almost all scenarios with SNP marker weighting further improved the prediction reliability between 0.5% and 12.7%. The highest improvement was achieved by weighing the SNP markers based on the 2pqû2 formula.

In vitro capsule or biofilm formation of Streptococcus uberis and bacteriological cure of bovine mastitis.

The relationship between the in vitro detected virulence factors biofilm and capsule formation of Streptococcus (S.) uberis isolates of clinical mastitis in dairy cows and the bacteriological cure rate after antibiotic therapy was investigated in order to better understand the importance of these virulence factors for the bacteriological cure rate. A total of 111 clinical mastitis (CM) cases were collected, in which S. uberis was bacteriologically detected. All mastitis cases were treated in accordance with the approval conditions of the antibiotic udder tubes used. Individual cow information including age, number of lactations, current lactation mastitis and antimicrobial treatment received was recorded. The microtiter plate test was used to detect biofilm formation and Anthony capsule staining was used to detect capsular capacity. Statistical analyses were performed to characterize the correlation between in vitro virulence factors and bacteriological cure (BC) rate. 30.5% (n=29) of the S. uberis isolates of bacteriologically cured cases and 34.5% (n=10) of the isolates of bacteriologically non-cured mastitis cases were found to have the ability to produce capsules in vitro. 70.7% (n=58) of the S. uberis isolates from bacteriologically cured mastitis cases had the ability to produce biofilm in vitro, whereas 58.6% (n=17) of the isolates of non-cured mastitis cases showed ability in producing biofilm. No correlation was found between the in vitro ability of S. uberis to form capsules and biofilms and the BC rate after antibiotic treatment of bovine mastitis. The present work has shown that the investigated in vitro virulence factors are not associated with the BC after antibiotic therapy. Further studies on the role of S. uberis virulence factors are needed to complete the missing knowledge on the difficulties in curing S. uberis mastitis. This study is of great clinical relevance since it enhances the understanding of the occurrence of BC in S. uberis mastitis cases. The investigated virulence factors are often addressed as possible reasons for therapy failure, although respective scientific studies are missing.

Assessment of potential treatments for clinical mastitis caused by Escherichia coli in cattle using clinical and biochemical markers

Assessment of potential treatments for clinical mastitis caused by Escherichia coli in cattle using clinical and biochemical markers

Proposal for the design of studies of intramammary antimicrobials to be conducted in Brazil for registration with the Ministry of Agriculture and Livestock

Before being made available to the Brazilian market, all veterinary medicines must be registered with the Ministry of Agriculture and Livestock after their efficacy, safety, and quality have been proven through clinical studies and scientific literature. Depending on the product class, the studies required by the regulatory authority must be carried out per Brazilian regulations and international reference guides. Some international organizations pursue the harmonization of these requirements, aiming at the mutual acceptance of studies conducted in different regions to facilitate international trade and reduce the use of animals in clinical research. Mastitis is one of the most prevalent and costly diseases in dairy cows, and it is associated with negative impacts on milk production and quality, cow welfare, and the profitability of the dairy industry. Normative Instruction No. 26/2009 determines a few rules for conducting studies for registering intramammary antimicrobials for cows with mastitis. However, the regulated sector and researchers report difficulties in following the recommendations of specific guidelines, which complement this regulation due to the peculiarities of Brazil’s production systems. This review article aims to provide subsidies and orientations that scientifically support the conduction and critical analysis of clinical studies proving the efficacy of intramammary products for treating clinical and subclinical mastitis in cows. Considering the need for scientific rigor in the studies, the recommendations available in international guidelines, and the need to adapt the protocols to the current situation of veterinary clinical research in Brazil, this document is intended to contribute to the internal harmonization of experimental protocols that support both the regulated sector and the regulatory authority.

Comparative Profiling of Milk Somatic Cells Proteomes Revealed Key Players in Mammary Immune Mechanisms During Mastitis in Tropical Sahiwal (Bos indicus) Cows.

Bovine mastitis poses a significant economic burden on the dairy industry worldwide. This pioneering proteomic study conducted a comparative profiling of milk somatic cell (SC) proteins contributing to mammary immune defense during subclinical and clinical mastitis (CM) in Sahiwal (Bos indicus) cows. Based on California mastitis test (CMT) scores, milk SC counts, differential leukocyte counts (DLCs), and bacteriological culture results, quarter milk SC samples were categorized into healthy (H), subclinical mastitis (SCM), and CM groups. Comparative proteome profiling of milk SCs was done using a label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic approach. The identified upregulated proteins in mastitis groups such as Vanin 2, Thioredoxin reductase-like selenoprotein T, Ceramidase, Lymphocyte antigen 75, Misshapen-like kinase 1 (MINK1), Thrombospondin 1, Macrophage scavenger receptor 1, Leupaxin, and Lipoamide acyltransferase, involved in immune responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed immune functions and pathways like antigen processing, complement cascades, extracellular matrix receptor interaction, efferocytosis, leukocyte migration, chemokine, peroxisome proliferator-activated receptors (PPARs), and transforming growth factor (TGF)-beta signaling. These findings provide essential information on proteomic profiling in milk SCs and contribute valuable insights into immune-related proteins regulated during mastitis in dairy cows. Further, validated proteins (Vanin 2, MINK1, and Thrombospondin 1) offer potential inflammatory biomarkers for early mastitis detection in dairy cows.

The investigation of SNP in SOCS2 gene and its effect on milk yield, fat, protein, and somatic cell count in Awassi ewes.

Livestock farmers face financial losses every year because milk yield and components are severely affected by udder diseases. These udder infections attract the immune response from the host and lead to the influx of neutrophils into milk to fight infection and thus the number of somatic cell count (SCC) is increased. The SCC value of milk could be used as an important indicator in detecting clinical mastitis in dairy animals. Also, the milk yield and milk quality (e.g. fat) are negatively affected by the increased SCC. The SCC is used to estimate the somatic cell score (SCS)of the milk, which is used as an indirect measure to detect subclinical mastitis. Therefore, the purpose of this study was to investigate the presence of a significant SNP rs868996547, on the suppressor of cytokine signaling 2 gene (SOCS2) which is related to milk yield and milk quality in Awassi sheep. In this study, milk production data was obtained from 210 healthy Awassi ewes with different parties and ages. The general linear model (GLM) process analysis of variance (ANOVA) was used to determine fixed effects on milk traits. The DNA extraction was done using a blood DNA extraction kit from Qiagen. To validate the presence of SNP a customized SNP detection developed by Thermofisher Scientific was used. The presence of the SNP in the SOCS2 gene was detected with genotypes (C/T, T/T, and C/C) and T being the mutated allele and it had a significant (p < 0.015) effect on the milk yield (p < 0,015;0.091), fat (p < 0,001;0,003), fat/protein ratio (p < 0.001;0,037) and log10SCC value (p < 0,006;0,015) of Awassi ewes. However, the protein, total solid, and lactose percentages in the wild type and the mutated ewes found having no significant difference (P > 0.05). Our result showed the increase in SCC or SCS of the milk significantly affected the milk yield and composition. Parity and age had significant effects on ewes' milk yield (p < 0.001). In conclusion, we investigated the presence of SOCS2 gene of Awassi ewes in the study flock and its effect on milk yield, fat, and somatic cell count, and the change in milk composition and milk yield because of SCC.

Thermographic assessment of mastitis progression in sahiwal cattle: Insights into the patterns in the natural course of infection

Mastitis is a global concern in the dairy sector, demanding innovative solutions for effective management for quality lifetime milk production. In this study, infrared thermography (IRT) as a non-invasive technology was integrated into routine farm activities for continuous health monitoring of animals. For 30 days, we systematically monitored the udder health status in 40 Sahiwal cows (160 quarters), employing IRT along with the California Mastitis Test (CMT). We also assessed somatic cell count (SCC), microbial identification, and milk quality parameters of representative samples. The thermal imaging data was analyzed, considering both backward propagation from the 0th day to the −10th day and forward propagation from the 0th day to the +10th day. Our findings revealed that on the 0th day, the mean temperatures of the udder surface skin temperature (USST) and teat skin surface temperature (TSST) exhibited differences (p < 0.05) between the quarters affected by sub-clinical mastitis (SCM) and clinical mastitis (CM) in comparison to the healthy quarters, with the highest degree of difference observed. The observed temperature differences between CM and SCM quarters compared to healthy ranged from 1.8 to 3.62 °C and 0.98 to 3.23 °C for USST, and from 1.68 to 3.16 °C and 0.56 to 2.32 °C for TSST, respectively. Furthermore, our observations indicated that both udder and teat quarters responded differently to mastitis. A temperature rise of 1.37 °C in SCM quarters and 1.75 °C in CM quarters was observed between the −10th and -8th day relative to day 0, with the increase being more pronounced in the morning hours. Also, a notable temperature surge occurred during the -2nd and -1st days relative to the 0th day. The log10SCC values and milk quality parameters significantly differed (p < 0.05) between mastitis-affected and healthy samples. In addition, Staphylococcus spp. was identified as the predominant mastitis-causing pathogen in the bacteriological identification conducted in this study. Therefore, IRT efficiently assesses the initiation point of udder infection in Sahiwal cows, aiding in effective udder health management.

Clinical mastitis in an Ettawa crossbreed ewe

Clinical mastitis in an Ettawa crossbreed ewe

Analysis of Inflammatory and Regulatory Cytokines in the Milk of Dairy Cows with Mastitis: A Comparative Study with Healthy Animals

ABSTRACT Bovine mastitis remains a major problem in the global dairy cattle industry. The acute invasion of udder by pathogens induces innate immune response as the first defence mechanism in subclinical and clinical mastitis. The aim of the study was to determine inflammatory and regulatory cytokines IL-2, IL-4, TGF-β1, IL-17A, beta-defensin 3 and IL-10 and their potential changes in milk of dairy cows with subclinical and clinical mastitis, and to compare the findings with healthy animals. Milk samples from 15 holstein Friesian breed cows were used in the study. Cows were divided into three groups based on their health status (5 healthy, 5 subclinical and 5 clinical animals). All samples were tested using immunohistochemistry to evaluate IL-2, IL-4, IL-10, IL17A, TGF-β1 and β-Def 3 proteins. Expression of all proteins was detected in all milk samples. High expression of IL-2, IL-4, IL17A, TGF-β1 was detected in healthy cows’ milk and in milk of cows with subclinical and clinical mastitis. However, expression of IL-10 and β-Def 3 in milk samples of healthy cows was significantly higher compared to the milk of cows with subclinical and clinical mastitis (p < .001). IL-10 and β-Def 3 can be considered as informative biomarkers in diagnosis of subclinical and clinical mastitis.

Phytochemical profile and evaluation of the antimicrobial activity of the crude hydroalcoholic extract of Cecropia pachystachya leaves

Cecropia pachysthacia belongs to the Urticaceae family, popularly known as embaúba, imbaúba, umbaúba, embaúva, sloth plant. This species is popularly used for the treatment of various pathologies, such as respiratory, hypertension, and blood glucose control, among others. The objective of the present work was to evaluate the phytochemical profile and the antimicrobial activity of the crude hydroalcoholic extract of Cecropia pachysthacia leaves. Phytochemical tests were performed to determine bioactive compounds with pharmacological action. Phytochemical screening demonstrated the presence of saponin, terpenes, volatile oils, glycosylated flavonoids, triterpenes, flavonoids, anglicones, and tannins. For the microbiological analysis, five genera were used, Escherichia coli, Klebsiella pneumoniae, Bacillus spp., Enterobacter aerogenes, Staphylococcus spp. isolates of bacteria from cows with clinical mastitis, from the bacteriothek of the Infectious Diseases Laboratory of the Federal Rural University of Pernambuco - UFRPE. The crude hydroalcoholic extract under study showed inhibitory activity against the tested isolates, resulting from the bacteriostatic effect of the extract components.

PSX-10 Genome-wide association studies for clinical mastitis in dairy cattle

Abstract Mastitis, the inflammation of the mammary gland caused by both gram-positive and gram-negative bacteria, causes the global dairy industry over $20 billion annually in losses due to changes in milk yield and quality. The goal of this study was to identify genetic variants, genes, and biological pathways that are associated with mastitis. We performed genome-wide association studies (GWAS) on 1,858 cows from two lactations with both medium (n = 87,493) and high density (n = 624,300) single nucleotide polymorphisms (SNP) using a single SNP mixed linear model implemented in GCTA software. A total of 288 SNPs across all evaluated models passed the 5% genome-wise false discovery rate threshold across all autosomes with emphasis on chromosomes 3, 14, 16, 12, 6, and 2. These SNPs were mapped to their corresponding genes using the bovine genome. Gene enrichment analysis was then performed to identify significant biological pathways associated with mastitis. The current study identified potential genes that are involved in the MAPK, C5a, and ion binding pathways, which have been previously associated with immune function and inflammation. Further validation and investigation are required to confirm the identified genes and pathways before implementation in breeding programs.

Risk factors of bovine clinical mastitis and antimicrobial resistance of mastitis-causing Escherichia coli in Shandong, China

Risk factors of bovine clinical mastitis and antimicrobial resistance of mastitis-causing Escherichia coli in Shandong, China

Prevalence of Clinical and Subclinical Cattle Mastitis and the Associated Risk Factors in Bomet County, Kenya

Prevalence of Clinical and Subclinical Cattle Mastitis and the Associated Risk Factors in Bomet County, Kenya