Small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.     

A new diagnostic indicator using concanavalin a low-affinity lactoferrin levels in mammary gland secretion in mastitic drying cows

We examined the effective diagnostic indicator using the concanavalin A (Con A) low-affinity lactoferrin (Lf) to mastitic drying cows. The concentrations of both Lf and Con A low-affinity Lf in mammary gland secretions (MGSs) were lower than normal MGSs at the early and middle dry periods and colostrums. On the other hand, the levels of Con A low-affinity Lf in MGSs increased following the appearance of mastitis symptoms, and decreased when the mastitic symptoms were cured. Moreover, IgG1 concentrations of colostrums decrease on the quarters where a high level of Con A low-affinity Lf was determined after the onset of dry period. These results suggest that this method could be used as a useful indicator to mastitic drying cows.     

Effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows

We examined combination therapy with both lactoferrin (Lf) and antibiotics on clinical mastitis due to Staphylococcus aureus (S.aureus) on drying cows. The clinical symptoms of mastitic quarters were cured 81% of combination therapeutic quarters at 7 days post injection (dpi). Moreover, most of mammary gland secretions (MGSs) in combination therapeutic quarters were normal at 7 days after parturition. In the quarters with combination therapy, S.aureus counts, Lf concentrations and content rate of concanavalin A (Con A) low-affinity Lf decreased and were lower than in the quarters treated with Lf or antibiotics alone. The mRNA expression of tumor necrosis factor alpha (TNFalpha) of the quarters with combination therapy also decreased and was lower than that of the Lf or antibiotics treated. The mRNA expression of pro-inflammatory cytokines and chemokines in bovine mammary gland epithelial lined cells (BMEC) stimulated with Lf were lower than those of Con A low-affinity Lf stimulated BMEC. Moreover, Lf showed an inhibitory effect to the induction of pro-inflammatory cytokine mRNA expression when co-stimulated with Lf and Con A low-affinity Lf. Nuclear factor kappa B (NFkappaB) activation was also induced with Con A low-affinity Lf, and the inhibitory effects of Lf were also confirmed on BMEC co-stimulated with Lf and Con A low-affinity Lf. These results indicated that the efficacy of combination therapy with antibiotics and Lf caused antibacterial effect of antibiotics and inhibition of pro-inflammatory cytokine and chemokine production with Lf via the inhibition of NFkappaB activation.     

Evaluation of the protective effect of bovine lactoferrin against lipopolysaccharides in a bovine mammary epithelial cell line

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.     

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.     

Concentrations and specific antibodies to staphylococcal enterotoxin-C and toxic shock syndrome toxin-1 in bovine mammary gland secretions,and inflammatory response to the intramammary inoculation of these toxins

To investigate the pathological role of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) in bovine mastitis, the production of SEs and TSST-1 was investigated in staphylococci isolated from 120 mammary gland secretions (MGS, 51 from no clinical sign-mammary glands and 69 from staphylococcal mastitic-mammary glands) collected from dairy farms where staphylococcal mastitis frequently occurred in Miyagi and Yamagata prefectures from 1997 to 1998. Concentrations of these toxins and specific antibody titers in each MGS were also measured. Furthermore, SEC and TSST-1 were inoculated into lactating mammary glands and inflammatory responses were analyzed. A high percentage of staphylococci including Staphylococcus aureus and coagulase-negative staphylococci isolated from both no clinical sign- and mastitic-MGS produced both SEC and/or TSST-1. The concentration of SEC increased with the severity of the mastitis, and was significantly higher (P<0.05) in acute mastitic-than in no clinical signs-MGS. Titers of specific antibodies to TSST-1 in MGS were significantly higher (P<0.05) than those to SEC, regardless of whether or not the cows were lactating or mastitic. Specific antibodies purified from MGS neutralized each toxin in vitro. A significant increase (P < 0.05) in somatic cell counts was induced by the intramammary inoculation of SEC but not TSST-1. These findings indicated that SEC rather than TSST-1 plays an important role in the pathology of staphylococcal bovine mastitis. The inflammatory activity of TSST-1 was probably neutralized by specific antibodies in MGS.     

Bovine lactoferrin binds to insulin-like growth factor-binding protein-3

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been shown to have IGF independent actions that appear to be mediated by specific IGFBP-3 binding proteins located on cell membranes. We show here using Western ligand blotting, a number of mammary membrane proteins that bind ¹²⁵I-labeled rhIGFBP-3. Immunoprecipitation studies demonstrated that the >70 kDa protein was identified from bovine mammary microsomes as bovine lactoferrin (bLf). In addition to being a secretory protein, Lf is tightly associated with cellular membranes. Labeled rhIGFBP-3 was shown to bind to commercially purchased and processed apo- or holo-human or bLf, but not bovine transferrin (bTf). Binding of [¹²⁵I]rhIGFBP-3 to other positively charged proteins was not detected nor was binding to rhIGFBP-5 or other mammary-secreted IGFBPs observed. Reciprocal specific binding of [¹²⁵I]bLf to rhIGFBP-3 was shown, but [¹²⁵I]bTf did not show binding to rhIGFBP-3. While [¹²⁵I]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [¹²⁵I]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED₅₀=3 μg/ml) or displaces (ED₅₀=1 mg/ml) bound [¹²⁵I]rhIGF-II from dot blotted rhIGFBP-3. In vitro studies with a bovine primary mammary epithelial cell culture showed that all-trans-retinoic acid stimulates the appearance of bovine IGFBP-3 and bLf in the conditioned media and that [¹²⁵I]rhIGFBP-3 could be utilized to detect conditioned media bLf. These findings reveal a novel role for bLf, binding to IGFBP-3 and perhaps disassociating IGFBP-3:IGF when in high concentration.     

Immunobiology of bovine mammary gland: apoptosis of somatic cells in milk during naturally occurring mastitis

Mastitis remains a major cause of economic losses in dairy herds. It is believed, that the enhancement of natural defense mechanisms in mammary gland may be helpful in the treatment of bovine mastitis. Our study was designed to assess the apoptosis of leukocytes isolated from bovine milk during subclinical staphylococcal mastitis. Milk samples were collected from cows naturally infected with one pathogen--Staphylococcus aureus and from animals with mastitis caused by several pathogens, including S. aureus. It has been determined that the rate of apoptosis was lower in mastitic milk, as compared with control samples, although varied significantly between groups. High percentage of apoptotic cells was detected in milk with high counts of pathogenic bacteria. In all groups the rate of apoptosis was dependent on the bacterial load, although various correlations were identified. Thus, it is postulated, that the rate of apoptosis of somatic cells in mastitic milk is related to the etiology of infection and is determined by the bacterial load.     

Cell free mitochondrial DNA in serum and milk associated with bovine mastitis: a pilot study

Mastitis is inflammation of mammary gland affecting all the species of domestic animals. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that bovine mastitis would be associated with increased cell free mitochondrial DNA (mtDNA) content in serum and milk. Milk and serum samples were collected from sub-clinical mastitic and normal animals. Mastitis was confirmed by California mastitis test and bacterial isolation. Oxidative stress, nitric oxide and inflammatory cytokines were also estimated. Real time polymerase chain reaction was conducted in serum and milk from sub-clinical mastitic animals and compared with healthy animals targeting the mtDNA genes cytochrome b. Mastitis animals showed higher oxidative stress markers and nitric oxide along with higher level of inflammatory cytokines. Cell free mtDNA was significantly higher in serum and milk of mastitic animals comparing to that of healthy control. The higher cell free relative mtDNA content in mastitis animals indicates injury to the mammary epithelial cells and thereby releasing the mtDNA in the milk and blood. This mtDNA may be a bio-marker of oxidative stress and tissue injury in bovine mastitis.     

The cytokines of bovine mammary gland: prospects for diagnosis and therapy

The lack of efficacy of conventional strategies for the maintenance of healthy udders in domestic cattle has prompted studies on the use of cytokines for this purpose. The adjuvant use of recombinant bovine cytokines, such as IL-2, IFN-gamma and TNF-alpha, in normal mammary gland, mobilizes innate and acquired immunity. However, stimulated immunity does not prevent or eradicate infection, particularly in the case of Staphylococcus aureus mastitis. Cytokines do, however, improve the bactericidal efficiency of certain antibiotics. The subtle and sensitive changes in the cytokine network of normal and mastitic bovine mammary gland may encourage the use of cytokines in the diagnosis and prognosis of udder health. Numerous studies support this hypothesis, and detection and monitoring of cytokines could become an important alternative management for udder health. The use of cytokines in the immunotherapy, diagnosis and prognosis of mastitis will grow with knowledge of the cytokine network in bovine mammary glands and the development of efficient cytokine diagnostic techniques.     

Bacteriostatic activity of bovine lactoferrin in mastitic milk

The antibacterial activity of milk against a virulent strain of Escherichia coli was investigated using milk fractions from normal or inflamed glands. Mastitic whey exhibited either bactericidal or bacteriostatic activities, depending on whether bacteria were enumerated by the pour plate technique or by surface plating onto sheep blood agar. The former activity was not due to lactoferrin (Lf), which never exerted bactericidal activity, even when assayed in distilled water. Milk whey ultrafiltrate (UF) (mol. wt. less than 5000 d) was used to assay the ability of normal and mastitic milk to support the antibacterial activities of Lf against a strain of E. coli. The addition of purified Lf to UF from mastitic whey resulted in bacteriostasis, whereas Lf was without effect in UF from normal whey. It was concluded that Lf can actually slow down the growth of Lf-sensitive bacteria during mastitis, provided that plasma exudation takes place.     

Lactoferrin interaction with retinoid signaling: cell growth and apoptosis in mammary cells

Lactoferrin (Lf) is a multifunctional iron-binding protein that was first identified in mammary secretions, but is synthesized by most mammalian tissues. The protein has a signal sequence that dictates secretion; it also has a nuclear localization sequence that facilitates entry into the cell nucleus. The mechanism of the latter action is currently unknown, but is thought to occur via a Lf receptor. Lactoferrin content of mammary tissue and secretions varies with developmental state; it is synthesized in mammary tissue at high levels during both pregnancy and involution, and during mammary infections. Using fluorescent (FITC)-labeled holo-bLf, we show that bovine primary epithelial cells and MCF-7 breast cancer cells do not translocate the exogenously added Lf to the nucleus after culture in serum free media (SFM). However, the supplementation of SFM with 1 μM all-trans retinoic acid (atRA) caused breast cancer cells to gain the capacity to take up labeled bLf into the cell nucleus. Primary bovine mammary cells (MeBo) exhibited similar capacity in culture. This suggests that in addition to Lf, one or more components modulated by atRA, are necessary for nuclear translocation to occur. Transfection experiments with atRA treated MCF-7 cells containing retinoic acid response element reporter constructs showed that the extracellular application of lactoferrin alters reporter gene expression. Lactoferrin increased a DR5 luciferase response element in a dose-dependent manner only when atRA was applied. Immunocytochemical markers for the cell cycle (Ki67) and apoptotic events (Caspase-3 and PARP-85) showed that lactoferrin alters the atRA-induced phenotype, blocking apoptosis and maintaining cell cycle activity in both MCF-7 and MeBo cells in the presence of 1 μM atRA. We propose that nuclear lactoferrin interacts with retinoic acid signaling pathways in cells and alters/blocks the signals so that cells remain in the cell cycle and/or do not enter the apoptotic pathway.     

Characteristics and epidemiologic genotyping of Staphylococcus aureus isolates from bovine mastitic milk in Hokkaido, Japan

Two hundred thirty one Staphylococcus aureus isolates from bovine mastitic milk were discriminated into 60 patterns and 16 lineages by pulsed-field gel electrophoresis (PFGE). The tested isolates were also investigated using coagulase and capsule serotyping and PCR for possession of genes that encode staphylococcal enterotoxins (sea to sei), enterotoxin-like toxins (selj to selr), and toxic shock syndrome toxin (tst). One hundred seventy three of the isolates (74.9%) possessed one or more toxin genes, while no egg-yolk factor was detected in most of them. The most common combinations of toxin genes possessed by the tested isolates were sec, seg, sei, sell, and tst, or seg and sei, or sec, seg, sei, sell, seln, and tst. Two hundred and ten of the isolates (91.0%) serotyped coagulase VI, and 207 of the isolates (89.6%) expressed serotype 5 or 8 capsules. These results suggested that isolates belonging to two major lineages have spread all over Hokkaido as bovine mastitic isolates. Additionally, no remarkable difference was recognized in the identification ratio of the isolates that belonged to the two major lineages between mastitis of subclinical origin and mastitis of clinical origin.     

Morphometric study of experimentally induced Staphylococcus bovis mastitis in the cow

Comparisons were made of bovine mammary tissue at 12 and 24 hours after 5,000 colony-forming units of Staphylococcus aureus 305 was injected into the mammary parenchyma with control tissue from the contralateral udder half. A mild form of staphylococcal mastitis was induced routinely. Generally, S aureus was found in udder tissue removed centrally from the site of injection. Varied degrees of tissue damage were found in infected quarters from alveolar epithelium without damage (similar to control) to extremely swollen epithelium which were typical of cells undergoing disruption. Infected tissues frequently displayed areas of swollen stroma and areas of diminished alveolar lumens. Cellular debris was often found in lumens, and neutrophils frequently filled the alveolar lumen in areas of greatest damage. Thus, a parenchymal injection of bacteria induced early forms of staphylococcal mastitis at the alveolar level.     

Dynamics of leukocytes and cytokines during experimentally induced Streptococcus uberis mastitis

Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.     

Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli

Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis.     

Predicted antigenic regions of Streptococcus uberis adhesion molecule (SUAM) are involved in adherence to and internalization into mammary epithelial cells

Streptococcus uberis is a significant cause of bovine mastitis throughout the world. Previous work from our laboratory demonstrated that S. uberis adhesion molecule (SUAM) is an important factor in adherence to and internalization of S. uberis into bovine mammary epithelial cells. Antibodies directed against SUAM significantly reduced bacterial adherence to and internalization into bovine mammary epithelial cells implying that SUAM is surface exposed. Objectives of this research were to: (1) predict surface exposed peptides, and (2) select peptide sequences for production of synthetic peptides with the final aim of evaluating their role in adherence and internalization and immunogenic potential. The Kyte/Doolittle hydropathicity prediction method; Chou/Fasman β-turn prediction method; and output from Coils, Paircoil and MultiCoil scores for prediction of secondary and tertiary structures were used. Prediction algorithms resulted in identification of five overlapping regions of the SUAM sequence with the most hydrophilic valleys and the highest peaks for β-turns. The five 15-mer SUAM epitopes selected by bioinformatic analysis were produced to evaluate the immunogenic value and pathogenic role of these putative domains. Peptides were bound to fluorescent latex beads, incubated with MAC-T bovine mammary epithelial cells, and internalization into MAC-T cells was evaluated using confocal laser and transmission electron microscopy. All peptides evaluated induced some degree of internalization of fluorescent beads into MAC-T cells; however, 2 peptides induced significantly more internalization of fluorescent beads than the other peptides evaluated. These peptides, designated III and IV, were located in the central region of SUAM, between two coiled-coil regions. Convalescent sera were tested against these biotinylated peptides for SUAM specific immune response using an indirect ELISA format. Among the 5 peptides evaluated, peptides I, II and V elicited significant serological response suggesting that the N-terminal region (peptide I), central region (peptide II) and C-terminal region (peptide V) are immunodominant epitopes of SUAM. Results will be useful to design immunotherapeutic tools based on immunodominant epitopes.     

Comparison of the response of bovine and human neutrophils to various stimuli.

Elastase release, oxidant production and cytoplasmic Ca2+ fluxes by bovine and human neutrophils were compared using sensitive kinetic assays on a photon-counting spectrofluorometer. The stimulants used were phorbol myristate acetate (PMA), cytochalasin B, zymosan opsonized with bovine complement (bOZ) or human complement (hOZ), calcium ionophore, formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A). The respiratory burst of bovine and human neutrophils was stimulated by PMA and OZ but not by cytochalasin B, or calcium ionophore. Con A weakly stimulated this response in human neutrophils but not bovine. FMLP stimulated the respiratory burst of human but not bovine neutrophils. For evaluation of elastase release, human neutrophils were pretreated with cytochalasin B for 5 min and then stimulated. Cytochalasin B alone did not stimulate elastase release from human neutrophils. Phorbol myristate acetate, calcium ionophore, hOZ, FMLP and Con A did stimulate human neutrophils pretreated with cytochalasin B to release elastase. Human serum OZ was also able to stimulate elastase release from human neutrophils not pretreated with cytochalasin B. Some bovine neutrophils released elastase in response to cytochalasin B alone. Those bovine neutrophils that did not release elastase in response to cytochalasin B alone released elastase when stimulated with Con A or calcium ionophore after cytochalasin B pretreatment. Bovine neutrophils did not release elastase in response to FMLP or PMA with or without cytochalasin B pretreatment, but did release elastase in response to bOZ alone. Total elastase activity of bovine neutrophils was determined to be about 50 times less than that of human neutrophils. Intracellular calcium fluxes were stimulated in human neutrophils by calcium ionophore, FMLP, hOZ and Con A but not by PMA or cytochalasin B. Bovine neutrophil calcium fluxes were stimulated by calcium ionophore, Con A and bOZ; cytochalasin B also stimulated bovine neutrophils to increase cytoplasmic calcium concentration. Cytoplasmic calcium fluxes were not stimulated in bovine neutrophils by PMA or FMLP. In summary, human and bovine neutrophils respond similarly to calcium ionophore and OZ, but differently to PMA, cytochalasin B, Con A and FMLP.     

Concentrations of IL-6 in Serum and Whey from Healthy and Mastitic Cows

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.     

Outcome prediction from the first examination in clinical mastitis using ultrasonography in dairy cows

The aim of this study was to examine whether ultrasonography can be used to predict the outcome of clinical mastitis in dairy cows. Forty‐seven mastitic quarters of Holstein‐Friesian cows were examined using ultrasonography at the time of the first examination. In mastitic mammary tissue, three sonographic signs indicating tissue abnormality were found: a hyperechoic spot in the parenchyma area, structural changes to the milk duct, and non‐homogeneous parenchyma. Logistic regression was used to evaluate whether the abnormal findings in the sonographic images can be used to predict the outcome of clinical mastitis. The outcomes of clinical mastitis were defined by the return, or failure to return, to marketable milk production. The sonogram finding of non‐homogeneous parenchyma in the first examination did predict the outcome of clinical mastitis, whereas the type of systemic symptoms (severe or moderate) was not a predictor in this regression model. Therefore, ultrasound examinations of mammary glands in the first examination could be a useful method for predicting outcome of clinical mastitis. There is an economic benefit if ultrasound examination in first examination helps in the decision of whether or not to treat the mastitic cows.