Bovine Udder Research Articles

In vitro ability of mastitis causing pathogens to form biofilms.

This Research Communication describes the study of in vitro biofilm formation of mastitis causing pathogens. Biofilms are communities of bacteria that are attached to a surface and to each other and are embedded in a self-produced matrix of extracellular polymeric substances. Biofilm formation is an important virulence factor that may result in recurrent or persistent udder infections and treatment failure through increased resistance to antibiotics and protection against host defences. In the present study 252 bacterial isolates from milk samples from bovine udder quarters with intramammary infections were examined with Congo Red agar (CRA) method and tube method (TM) for their ability to form biofilms. Both tests revealed a high number of biofilm-positive strains. Literature reports that the cure rates for Staphylococcus aureus infected udders are lower (27%) in comparison to cure rates of Streptococcus uberis (64-81%) or coagulase-negative staphylococci (CNS) mastitis (80-90%). The findings of the present study suggest that biofilm formation is not the main factor for the differences in cure rates of the various bacteria genera, because all tested pathogen groups showed a similarly high proportion of biofilm formation. Further research is needed to detect microbial biofilms on bovine udder epithelia.

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.

SHORT-COMMUNICATION Evaluation of perfused bovine udder for gene expression studies in dairy cows

Intramammary infections are one of the main causes of productivity loss in dairy cows. To better understand the immune system response and to avoid the use of live animals, we validated the use of isolated bovine udder as an ex situ model. Six mammary glands were collected from cows ready for culling. Three udders were perfused with Tyrode's solution and three were not-perfused. During six hours, we collected perfusate samples for biochemical analysis. We also collected alveolar and teat canal tissue to evaluate gene expression. The biochemical parameters indicated that the perfused udders remained viable for the entire period of the experiment. A real-time polymerase chain reaction showed an increase in 18S rRNA gene expression in the alveolar tissue at 3 and 4 h after perfusion. There was also an increase in the Ubiquitin gene in the teat canal from not-perfused udders at 1, 3, and 4 h after slaughter. In general, gene expression was stable during the experiment. Our results indicated that the isolated perfused bovine udder model is appropriate for genetic studies, opening a new perspective in animal experimentation methods.

Persistence of a dominant bovine lineage of group B Streptococcus reveals genomic signatures of host adaptation.

Group B Streptococcus (GBS) is a host-generalist species, most notably causing disease in humans and cattle. However, the differential adaptation of GBS to its two main hosts, and the risk of animal to human infection remain poorly understood. Despite improvements in control measures across Europe, GBS is still one of the main causative agents of bovine mastitis in Portugal. Here, by whole-genome analysis of 150 bovine GBS isolates we discovered that a single CC61 clone is spreading throughout Portuguese herds since at least the early 1990s, having virtually replaced the previous GBS population. Mutations within an iron/manganese transporter were independently acquired by all of the CC61 isolates, underlining a key adaptive strategy to persist in the bovine host. Lateral transfer of bacteriocin production and antibiotic resistance genes also underscored the contribution of the microbial ecology and genetic pool within the bovine udder environment to the success of this clone. Compared to strains of human origin, GBS evolves twice as fast in bovines and undergoes recurrent pseudogenizations of human-adapted traits. Our work provides new insights into the potentially irreversible adaptation of GBS to the bovine environment.

Detection of the biofilm component polysaccharide intercellular adhesin in Staphylococcus aureus infected cow udders

Biofilms are communities of microorganisms embedded in a self-produced extracellular matrix made up of polymeric substances. They reduce the effects of antibiotics and allow the microorganisms to evade the innate immune system. This can lead to persistent or recurrent infections. In dairy cow herds, mastitis is a serious problem. The present study aimed to investigate the occurrence of biofilms in the udders of dairy cows infected with Staphylococcus (S.) aureus, because biofilms may affect the response to treatment of bovine mastitis. Immunofluorescence staining of polysaccharide intercellular adhesin (PIA), a component of S. aureus biofilms, was carried out based on swabs taken from different areas of S. aureus infected udders. We were able to demonstrate the presence of PIA in S. aureus infected bovine udders. However, the applied method is invasive and therefore only really suitable for scientific research and not for clinical diagnosis.

Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese.

Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Due to its zoonotic potential, control of S. aureus is not only of great economic importance in the dairy industry but also a significant public health concern. The aim of this study was to decipher the potential of bovine udder associated S. aureus as reservoir for S. aureus contamination in dairy production and processing. From 18 farms, delivering their milk to an alpine dairy plant for the production of smeared semi-hard and hard cheese. one thousand hundred seventy six one thousand hundred seventy six quarter milk (QM) samples of all cows in lactation (n = 294) and representative samples form bulk tank milk (BTM) of all farms were surveyed for coagulase positive (CPS) and coagulase negative Staphylococci (CNS). Furthermore, samples from different steps of the cheese manufacturing process were tested for CPS and CNS. As revealed by chemometric-assisted FTIR spectroscopy and molecular subtyping (spa typing and multi locus sequence typing), dairy cattle represent indeed an important, yet underreported, entrance point of S. aureus into the dairy chain. Our data clearly show that certain S. aureus subtypes are present in primary production as well as in the cheese processing at the dairy plant. However, although a considerable diversity of S. aureus subtypes was observed in QM and BTM at the farms, only certain S. aureus subtypes were able to enter and persist in the cheese manufacturing at the dairy plant and could be isolated from cheese until day 14 of ripening. Farm strains belonging to the FTIR cluster B1 and B3, which show genetic characteristics (t2953, ST8, enterotoxin profile: sea/sed/sej) of the recently described S. aureus genotype B, most successfully contaminated the cheese production at the dairy plant. Thus, our study fosters the hypothesis that genotype B S. aureus represent a specific challenge in control of S. aureus in the dairy chain that requires effective clearance strategies and hygienic measures already in primary production to avoid a potential transfer of enterotoxic strains or enterotoxins into the dairy processing and the final retail product.

Genomic content typifying a prevalent clade of bovine mastitis-associated Escherichia coli.

E. coli represents a heterogeneous population with capabilities to cause disease in several anatomical sites. Among sites that can be colonised is the bovine mammary gland (udder) and a distinct class of mammary pathogenic E. coli (MPEC) has been proposed. MPEC are the principle causative agents of bovine mastitis in well-managed dairy farms, costing producers in the European Union an estimated €2 billion per year. Despite the economic impact, and the threat this disease presents to small and medium sized dairy farmers, the factors which mediate the ability for E. coli to thrive in bovine mammary tissue remain poorly elucidated. Strains belonging to E. coli phylogroup A are most frequently isolated from mastitis. In this paper, we apply a population level genomic analysis to this group of E. coli to uncover genomic signatures of mammary infectivity. Through a robust statistical analysis, we show that not all strains of E. coli are equally likely to cause mastitis, and those that do possess specific gene content that may promote their adaptation and survival in the bovine udder. Through a pan-genomic analysis, we identify just three genetic loci which are ubiquitous in MPEC, but appear dispensable for E. coli from other niches.

Molecular characterization and in-silico analysis of complete coding sequence of bubaline mLYS

The role of immune-relevant genes involved in disease biology as well as innate and adaptive immunity is at the centre stage while exploring the genetic basis of disease occurrence. The macrophage expressed lysozyme gene product contributes to innate immunity by cleaving the ß- 1,4 linkage of bacterial peptidoglycan in phagocytosis. Higher specific activity observed in bubaline milk/ serum lysozymes over their bovine counterparts is expected to contribute higher resistances to diseases in general and mastitis in particular. To explore the reasons for higher activity in terms of sequence variations, macrophage expressed lysozyme cDNA was synthesized, cloned and sequenced. The 593 bp mRNA sequence revealed a 444 bp (27nt to 470nt) ORF bearing usual start codon ATG and end codon TAA with GC content of 46 % coding for precursor polypeptide of 147 amino acids. The comparative sequence analysis of cattle and buffalo revealed the difference at 10 places leading to 3 non-synonymous (5th, 116th and 142th) substitutions which did not affect the predicted 3D structure. The mLYS polypeptide had signal sequence (1st to 18th residue) and the mature peptide had two lysozyme catalytic sites, three Ca++ binding sites as well as eleven catalytic clefts prominent among the conserved domains. Secondly, bubaline mLYS was phylogenetically closer to the abomasum type than mammary gland type. The high similarity in the coding sequences and predicted structure suggested that bubaline lysozyme gene if hyper-expressed either in native or recombinant form in bovine udder may result in better udder health.

EC-18 (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) Exhibits the Immune Regulatory Role through HSC Differentiation of Bone Marrow Cells in Mice

EC-18 (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) was originally isolated as a component of an extract from deer antler is traditionally used as an oriental medicine for hematopoiesis. A trace of MADG (monoacetyldiacylglyceride) could be detected in the deer antler, animal tissue, seed oils and bovine udder, which is identical to its natural source of MADG was chemically synthesized with glycerol, palmitic acid and linoleic acid. Previous study described that EC-18 has effect on the proliferation of hematopoietic stem cells (HSCs) (Biol. Pharm. Bull. 2004, 27(7): 1121-1125). Successively, we investigated the biological role of EC-18 in the differentiation of bone marrow cells into progenitor cell population in mice. EC-18 was administered daily for 1, 5 and 15 days to 6 week-aged C57BL/6 mice. Bone marrow cells of EC-18 administrated mice were collected from femurs and tibiae. Cell population was analyzed by FACs. As results, the total number of bone marrow cells was increased in EC-18 administered mice. To identify hematopoietic lineage cell population, we used lineage cocktail antibody kit including anti-mouse CD3, CD11b, CD45R, Gr-1 and TER-119. The lineages negative population contains HSCs which can self-renew and generate into all lineages of the hematopoietic system. The lineage negative cells significantly increased in time dependent manner in the EC-18-daily administrated mice. Cell population between myeloid progenitor (Lin- Sca1- Kit+) and common lymphoid progenitors (Lin- Sca1+ Kit+ IL-7R-α+) were analyzed using the antibody for Sca-1, c-Kit, IL-7R-α. The data showed that the population of myeloid progenitor cells was markedly increased rather than common lymphoid progenitor cells. In myeloid progenitor cell population, the cell population of megakaryocyte/erythrocyte progenitors (MEP) and granulocyte-monocyte progenitors (GMP) also elevated significantly. Taken together, EC-18 may have a potential role in the HSC differentiation and could be used as a therapeutic agent for anemia and neutropenia. DisclosuresNo relevant conflicts of interest to declare.

Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates to human cells, suggesting for the first time the ability of bovine isolates to infect human cells.

Effects of Rhodomyrtus tomentosa Leaf Extract on Staphylococcal Adhesion and Invasion in Bovine Udder Epidermal Tissue Model.

Bovine mastitis is one of the most important infectious diseases in dairy herds, and staphylococci are the most important etiologic agents of this disease. Antibiotics and chemical agents used in livestock for prevention and cure of the disease can accumulate in milk and give rise to food safety concerns. Rhodomyrtus tomentosa leaf extract was studied as an alternative approach to reduce the bacterial infections. The ethanolic extract of this plant demonstrated antibacterial activity with minimum inhibitory concentration (MIC) values as low as 16–64 μg/mL against staphylococcal isolates. In addition, the extract had an effect on the bacterial cell surface properties by increasing its hydrophobicity in a concentration dependent manner. To further extend the antibacterial efficacy, silver nanoparticles synthesized with the extract, a pure rhodomyrtone, and liposomal encapsulated rhodomyrtone were applied and their inhibitory effects on bacterial adhesion and invasion were determined by ex vivo study in a bovine udder epidermal tissue model. These agents exerted remarkable antibacterial activity against staphylococci and decreased the adhesion of the bacterial cells to the tissues. These results supported that R. tomentosa ethanolic extract could be applied as an alternative agent for bovine udder care in dairy farms.

Correction of data truncation artifacts in differential phase contrast (DPC) tomosynthesis imaging

The use of grating based Talbot–Lau interferometry permits the acquisition of differential phase contrast (DPC) imaging with a conventional medical x-ray source and detector. However, due to the limited area of the gratings, limited area of the detector, or both, data truncation image artifacts are often observed in tomographic DPC acquisitions and reconstructions, such as tomosynthesis (limited-angle tomography). When data are truncated in the conventional x-ray absorption tomosynthesis imaging, a variety of methods have been developed to mitigate the truncation artifacts. However, the same strategies used to mitigate absorption truncation artifacts do not yield satisfactory reconstruction results in DPC tomosynthesis reconstruction. In this work, several new methods have been proposed to mitigate data truncation artifacts in a DPC tomosynthesis system. The proposed methods have been validated using experimental data of a mammography accreditation phantom, a bovine udder, as well as several human cadaver breast specimens using a bench-top DPC imaging system at our facility.

Coagulase‐Negative Staphylococci Collected from Bovine Milk: Species and Antimicrobial Gene Diversity

AbstractThe aim of this study was to examine the genetically mediated antimicrobial resistance in 94 coagulase‐negative staphylococcal (CNS) milk isolates (buffalo, n = 88, and cow, n = 6), and to determine whether antimicrobial resistance profiles differed between bacterial species. Our analysis of 94 CNS isolates from milk confirmed the well‐established multiresistant character of staphylococci in the dairy setting. Resistance against oxacillin, ciprofloxacin and cefoxitin was most frequently observed. Eleven CNS species isolated from buffalo's and cow's milk samples were 100% sensitive to gentamicin, erythromycin, clindamycin and ciprofloxacin. Resistance to oxacillin was attributed to the mecA gene in 44.7% of the oxacillin‐resistant isolates. The mecA gene was detected in Staphylococcus intermedius, epidermidis, hominis, hyicus, caprae, sciuri, lugdunensis and xylosus while totally absent in chromogenes, simulans and lentus. Of the 11 CNS species, S. epidermidis, S. lugdunensis, S. hominis, S. xylosus and S. intermedius were the only species that exhibited multiple resistance.Practical ApplicationsStudying coagulase‐negative staphylococci (CNS) at the species level can provide valuable information about species‐specific differences that can be vital data to prevent the dissemination of antimicrobial‐resistant genes and resistant pathogens to the community. CNS have long been regarded as pathogenic, but their important role as colonizers of the bovine udder has been recognized and studied in recent years. CNS have become increasingly resistant to multiple antibiotics. In recent years, increasing numbers of reports have shown that the mecA gene is present in CNS strains, including hospital‐acquired infections, community‐associated methicillin‐resistant Staphylococcus aureus infections, animal epidermis, beaches and public transportation systems. Therefore, it is highly important to detect the mecA gene as a marker to investigate antibiotic resistance in milk that may represent a serious health and economic concern.

Complete genome sequence and comparative genome analysis of Streptococcus parauberis KCTC11980

Streptococcus parauberis is recognized as an alpha-hemolytic gram-positive bacterial pathogen associated with bovine udder mastitis. Recently, S. parauberis was identified as the major pathogen of olive flounder (Paralichthys olivaceus) streptococcosis in Northeast Asia, a disease that causes severe economic losses by triggering massive olive flounder mortality. In this study, the complete genome sequence of S. parauberis KCTC11980 was determined and analyzed. S. parauberis KCTC11980 harbors a single chromosome of 2,179,251 bp containing 2281 predicted coding sequences and featuring an average G+C content of 35.65 %. Dot-plot analysis of the entire genomic sequence and phylogenetic analysis of the 16S rRNA gene revealed a close evolutionary relationship between S. parauberis KCTC11980 and S. uberis. Our findings could serve as a basis for understanding the pathogen-host relationship and developing new prophylactic and therapeutic strategies against fish streptococcosis. This study also enhances our understanding of the physiology and evolution of a key representative of the Streptococcaceae family.

Reduction of Lactobacillus in the milks of cows with subclinical mastitis.

Clinical and subclinical bovine mastitis are the most frequent diseases encountered on dairy farms worldwide, which cause significant economic loss and veterinary cost. The mastitic disease status is associated with increases in both milk bacterial pathogens and somatic cell count (SCC). Although it is well established that the mastitic pathogens generally correlate with the milk SCC, to our knowledge, the correlation between the probiotic genus, Lactobacillus, and the mastitic causative bacteria and SCC have not been determined previously. Thus, in this study, milk samples from 12 mild and 28 severe subclinical mastitic dairy cows were collected from the same farm. The overall milk bacterial load was quantified with the total plate count method. The Lactobacillus genus and 4 common clinical and subclinical mastitic pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Trueperella pyogenes) in the sampled milk were enumerated by quantitative PCR. Mild and severe subclinical mastitic samples were distinctly separated on the principal component analysis score plot generated based on the quantities of these 5 target bacteria, suggesting that clear differences existed in the microbiological composition between the two sample groups. Based on comparison with the pairwise Mann-Whitney test, the mild subclinical mastitic dairy cows had a significantly higher amounts of lactobacilli (P=0.0175), but lower E. coli (P=0.0002), S. aureus (P<0.0001), S. agalactiae (P=0.0001) and T. pyogenes (P=0.0044) quantities, while an opposite trend occurred in the severe subclinical mastitic group. The negative correlation between Lactobacillus and the pathogenic bacteria, as well as the SCC, was confirmed with Spearman correlation analysis. Data generated from the current study may hint to a close relationship between Lactobacillus and the health of bovine udder.

Genome Sequences of Two Bovine Mastitis-Causing Escherichia coli Strains.

Escherichia coli is one of the main pathogenic agents causing inflammatory infections in the bovine udder. Here, we report the draft genome sequences of two strains isolated from different cases of clinical mastitis.

Fluorometric determination of free and total isocitrate in bovine milk

Isocitrate is an intermediate metabolite in the citric acid cycle found both inside the mitochondria as well as outside in the cytosolic shunt. Oxidation of isocitrate is believed to deliver large fractions of energy [i.e., reducing equivalents (NADPH) in the bovine udder] used for fatty acid and cholesterol synthesis. This study describes a new analytical method for determination of free and total isocitrate in bovine milk where time-consuming pretreatment of the sample is not necessary. Methods for estimation of both total isocitrate and free isocitrate are described, the difference being the esterified or even lactonized isocitrate. On average, 20% (6–27%) of cow milk isocitrate was esterified and free isocitrate correlated significantly with total isocitrate (r=0.98). The present fluorometric determination correlated well with the traditional spectrophotometric determination of isocitrate. Milk samples from Danish Holstein cows (984) contained significantly less isocitrate than milk from Danish Jersey cows (760; i.e., 0.134 vs. 0.211mmol/L). Isocitrate in milk is correlated to milk protein, fat, and citrate, and it is speculated, based on biochemistry, former studies, and the present, that isocitrate may reflect the energy situation in the mammary gland. The use of isocitrate as a biomarker of the energy status in the dairy cow is warranted.

A genome-wide association study reveals a QTL influencing caudal supernumerary teats in Holstein cattle.

Supernumerary teats represent a common abnormality of the bovine udder. A genome-wide association study was performed based on the proportion of the occurrence of supernumerary teats in the daughters of 1097 Holstein bulls. The heritability of caudal supernumerary teats without mammary gland in this study was 0.604. The largest proportion of the heritability was attributable to BTA 20. The strongest evidence for association was with five SNPs on chromosome 20, referred to as a QTL. The mode of inheritance at this QTL was dominant. These findings reveal that the occurrence of caudal supernumerary teats without mammary gland in Holstein cattle is influenced by a QTL on chromosome 20 and a polygenic part. The data support the high potential of the SNPs in the QTL region as markers for breeding against caudal supernumerary teats.

Intramammary administration of platelet concentrate as an unconventional therapy in bovine mastitis: First clinical application

Bovine udder infections induce a variety of changes in gene expression of different growth factors that may suggest their possible role in glandular tissue protection or repair processes. Growth factors and also chemokines and cytokines may act synergistically to increase the infiltration of neutrophils and macrophages to promote angiogenesis, fibroplasia, matrix deposition, and, ultimately, re-epithelialization. Considering the vast applications, typically in human medicine, of platelet concentrate (PC) and its ease of preparation, the aim of our study was to evaluate an alternative therapy to stimulate the regeneration of glandular tissue, administering a concentration in excess of the growth factors contained in the PC. In each one of the 3 farms examined in the trial, PC was prepared from donor cows in good health, free from infections, and with no records of medications administered during the previous 2 mo. The platelet produced in one farm was used only for treating the cows of the same farm in a heterologous way. A total of 229 mastitic quarters were divided in 3 groups: antibiotic group (treated with intramammary antibiotic), antibiotic and PC group (treated intramammarily with antibiotics in association with PC), and PC group (treated with intramammary PC alone). The diagnosis of mastitis was based on somatic cell count and bacteriological evaluation of the milk from the affected quarter. Platelet concentrate, alone or in association with antibiotic, was used for 3 consecutive days as an unconventional therapy in bovine acute and chronic mastitis. Our data show that the associated action of antibiotic and PC performed significantly better than the antibiotic alone, either for the recovery of the affected mammary quarters or for somatic cell count reduction. In the same way, the association antibiotic plus PC showed significantly fewer relapses compared with the antibiotic alone, either for acute or chronic mastitis. The treatment with only PC did not show statistically significant differences compared with both antibiotic alone or associated treatment for acute mastitis, and it was better than the use of only antibiotic for chronic mastitis. Our results show that PC alone may be useful for a quick resolution of the inflammatory response, playing a role in limiting the tissue damage to the mammary gland parenchyma and reducing the recurrence rates.