lactoferrin and Mastitis--Bovine

Mastitis, a symptom of inflammation in mammary tissue by infection with various kinds of bacteria, causes huge economic losses in the milk industry. One of the popular methods for treatment of mastitis is antibiotics, although this prohibits milk shipping and sometimes causes resistant microbes. Therefore, a new strategy to treat mastitis without antibiotics is eagerly required around the world. Antimicrobial factors belong to innate immunity and can start their function extremely early after bacterial stimulation. These factors have antimicrobial activity for a broad spectrum of bacteria. Elucidation of causal mechanisms and functions of antimicrobial factors in the mammary gland is thought to result in suitable methods for prevention and treatment of mastitis. Therefore, this review introduces traits of some antimicrobial factors and the mechanisms for expressing, producing and secreting them in the mammary gland. For antimicrobial factors, lingual antimicrobial peptide (LAP), S100A7, cathelicidin and lactoferrin are controlled in different sites and different time courses, suggesting that antimicrobial factors play different roles for local defense against bacterial infection in the mammary gland. These findings will contribute to the development of prevention and treatment methods for mastitis.

Topics: Animals; Antimicrobial Cationic Peptides; beta-Defensins; Cathelicidins; Cattle; Female; Humans; Lactoferrin; Mammary Glands, Human; Mastitis, Bovine; S100 Calcium Binding Protein A7; S100 Proteins

Lactoferrin is a multifunctional, iron-binding glycoprotein found in milk and other exocrine secretions. Lactoferrin in milk plays vital roles in the healthy development of newborn mammals, and is also an innate resistance factor involved in the prevention of mammary gland infection by microorganisms. Inflammation of the udder because of bacterial infection is referred to as mastitis. There have been many investigations into the relationships between lactoferrin and mastitis, which fall into several categories. The main categories are fluctuations in the lactoferrin concentration of milk, lactoferrin activity against mastitis pathogens, elucidation of the processes underlying the onset of mastitis, participation of lactoferrin in the immune system, and utilization of lactoferrin in mastitis treatment and prevention. This minireview describes lactoferrin research concerning bovine mastitis. In the 1970s, many researchers reported that the lactoferrin concentration fluctuates in milk from cows with mastitis. From the late 1980s, many studies clarified the infection-defense mechanism in the udder and the contribution of lactoferrin to the immune system. After the year 2000, the processes underlying the onset of mastitis were elucidated in vivo and in vitro, and lactoferrin was applied for the treatment and prevention of mastitis.

Topics: Animals; Anti-Infective Agents; Cattle; Female; Lactoferrin; Mastitis, Bovine

The widespread use of antibiotics has lead to the increased presence of pathogens that are less susceptible to their antibacterial effect. Lactoferrin (Lf) is naturally produced by the mammary gland. Lactoferrin is the main whey protein in human milk and is also present in cow's milk but at a much lower concentration than in human milk. This protein appears to have many biological functions, including antibacterial and antiinflammatory activities. The best-known effect of Lf is to bind iron that is essential for bacterial growth. However, the cationic nature of this protein also appears to be important for the antimicrobial activity of this protein. Lactoferrin has a weak antibacterial effect when used alone, but interestingly, Lf appears much more effective when used at low concentration in combination with several antibiotics. The most striking observation is that Lf increases the inhibitory activity of penicillin up to 4-fold in most penicillin-susceptible Staphylococcus aureus strains, whereas this increase was 4- to 16-fold in penicillin-resistant strains. Indeed, Lf reduces beta-lactamase activity in S. aureus strains producing this enzyme. Transcription of beta-lactamase gene is dramatically repressed in the presence of Lf. We evaluated the efficacy of intramammary treatments containing penicillin G or bovine Lf (bLf), or both, to cure chronic mastitis caused by a clinical isolate of S. aureus highly resistant to beta-lactam antibiotics. In a first trial, mastitis was induced in lactating cows by injecting a low dose of S. aureus through the teat canal of all quarters. Bacterial cure rate was null for control quarters, 11.1% for bLf, 9.1% for penicillin, and 45.5% for the combination of bLf and penicillin. A second trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. Quarters were treated with 100,000 IU of penicillin G with or without 250 mg of bLf for 7 d. Bacterial cure rate was greater for the bLf + penicillin combination (33.3%) compared with penicillin alone (12.5%). In conclusion, bLf added to penicillin is an effective combination for the treatment of stable S. aureus infections resistant to beta-lactam antibiotics.

Topics: Animals; Anti-Bacterial Agents; beta-Lactam Resistance; Cattle; Drug Synergism; Enterobacteriaceae; Female; Immune System; Lactoferrin; Mastitis, Bovine; Penicillin G; Staphylococcal Infections; Staphylococcus

In the mammary gland of cattle there is a complex defense system of non-specific and specific reactions available preventing the invasion of pathogenic bacteria. Most infections occur via the teat canal, so teat canal keratin (SKK) is of particular importance in non-specific defense of the gland. The SKK serves as a physical barrier, and bacteriostatic and/or bactericidal effects of SKK lipids and proteins against certain mastitis bacteria could be demonstrated. By increasing the concentrations of lactoferrin and lysozyme in milk a reduction of mastitis frequencies could be observed. However, those high concentrations in the proteins occur only during the dry period of the cow. An improvement of the mastitis situation would also appear possible by increasing phagocytosis. The numerous trials intended to reduce mastitis by improving specific protection showed no significant success. Therefore, the most successful and cheapest means to achieve udder health remains the strict and consistent hygiene of housing, animals and mammary glands.

Topics: Animals; Bacterial Infections; Cattle; Female; Keratins; Lactoferrin; Lipids; Mammary Glands, Animal; Mastitis, Bovine; Milk; Milk Proteins; Muramidase; Phagocytosis

Frequency of new intramammary infection is greatest during early involution, decreases during middle stages, and then increases prepartum. Penetrability of the teat canal, antibacterial properties of keratin, bacterial adherence, and epithelial sensitivity to toxins play a role in resistance. Leukocytes phagocytose bacteria and regulate expression of immune mechanisms, although their function is compromised during certain stages of involution. These cells increase to millions per milliliter as involution progresses and then decrease prepartum. Macrophages predominate in lacteal secretions, followed by lymphocytes and neutrophils. Lactoferrin, a major whey protein and iron chelator, is also associated with resistance to infection during the nonlactating period and may have immunomodulatory properties. Lacteal immunoglobulins increase throughout involution peaking prepartum and function by opsonizing bacteria, neutralizing toxins, and preventing bacterial adherence. Immunoglobulins are derived from blood or are produced locally by plasma cells present in the subepithelial mammary stroma. Plasma cells, lymphoid cells, and other protective leukocytes present in teat end tissues accumulate during infection and concentrations increase in response to local antigenic stimulation. Various aspects of the mammary immune system are compromised during periods of functional transition. Thus, vaccination, immunostimulation, accelerated involution, and intramammary devices are some methods now being tested to amplify local immunity and protect the gland from bacterial infection.

Topics: Animals; Cattle; Female; Immunoglobulins; Lactation; Lactoferrin; Leukocytes; Macrophages; Mammary Glands, Animal; Mastitis, Bovine; Milk; Neutrophils; Phagocytosis; Pregnancy

Understanding basic defenses of the udder is instrumental in developing measures to prevent mastitis. The teat canal is the first defense against pathogens, providing a physical barrier and antimicrobial substances. When bacteria breach the teat canal, milk leukocytes provide a second defense by ingesting pathogens. Intramammary devices have been used experimentally to increase leukocyte numbers and to enhance destruction of bacteria. Milk antibodies opsonize and lyse bacteria, neutralize toxins, and prevent adhesion to tissue. Vaccinating cows against mastitis generally has been unsuccessful; however, immunization is useful in controlling specific bacterial strains. Antibody-producing plasma cells preferentially accumulate in internal teat end tissues. Because bacteria contact these tissues to reach milk-producing areas of the udder, an immunostimulant to enhance locally the protective nature of plasma cells may decrease the occurrence of infection.

Topics: Animals; Antibodies; Antibody Formation; Cattle; Female; Immunity, Cellular; Immunization; Immunoglobulins; Lactation; Lactoferrin; Leukocytes; Lymphocytes; Macrophages; Mammary Glands, Animal; Mastitis, Bovine; Milk; Neutrophils; Pregnancy

Topics: Animals; Apoproteins; Cattle; Citrates; Female; Immunity; Klebsiella pneumoniae; Lactation; Lactoferrin; Lactoglobulins; Mammary Glands, Animal; Mastitis, Bovine; Pregnancy; Time Factors

Topics: Animals; Cattle; Colostrum; Female; Hexosaminidases; Hexosyltransferases; Immunoglobulins; Lactalbumin; Lactation; Lactoferrin; Lactoperoxidase; Leukocytes; Mammary Glands, Animal; Mastitis, Bovine; Milk; Milk Proteins; Muramidase; Pregnancy

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.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cephalosporins; Concanavalin A; Cytokines; Drug Therapy, Combination; Electrophoresis, Gel, Two-Dimensional; Female; Lactoferrin; Mastitis, Bovine; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus

The effect of bovine lactoferrin (Lf) was studied in experimental Escherichia coli mastitis, using enrofloxacin as a comparator. Mastitis was induced in six clinically healthy primiparous dairy cows by infusing 1500 colony-forming units of E. coli into a single udder quarter. The challenge was repeated into a contralateral quarter of the same cows 3 weeks later. At the first challenge, three cows were treated with 1.5 g of bovine lactoferrin intramammarily three times (12, 20 and 36 h postchallenge, PC), and the other three cows received 5 mg/kg of enrofloxacin (Baytril) parenterally (12, 36 and 60 h PC). Flunixin meglumine (2.2 mg/kg) was administered to all cows twice at 24-h intervals. During the second challenge, the treatments for the two groups were reversed. Intramammary challenge with E. coli produced clinical mastitis in all cows, but the severity of the disease varied markedly. No statistically significant differences between treatment groups were observed in clinical signs such as rectal temperature, rumen motility and general attitude. Milk somatic cell count, daily milk yield and bacterial counts in cows treated with Lf and those receiving enrofloxacin also did not differ significantly. However, a trend for a more rapid elimination of bacteria was seen in the cows treated with enrofloxacin. Milk NAGase activity also decreased significantly faster in the group treated with enrofloxacin. The concentration of lipopolysaccharide in milk compared with the number of bacteria was significantly lower in Lf than in enrofloxacin-treated cows (20 h PC).

Topics: Administration, Oral; Animals; Cattle; Dairying; Enrofloxacin; Escherichia coli Infections; Female; Fluoroquinolones; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Quinolones; Severity of Illness Index; Treatment Outcome

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.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cattle Diseases; Complement C3; Female; Lactation; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Staphylococcal Infections; Staphylococcus; Time Factors

Lactoferrin (LTF) is an iron-binding glycoprotein found in milk and other exocrine secretion with antibacterial activity proposed as an alternative to mastitis treatment or prevention. LTF has been proposed as a candidate gene for mastitis resistance selection. The aim of this paper was to assess LTF promotor to explore variations with potential association to mastitis resistance in dairy cows from Honduras.. A resequencing of promotor and Exon I of LTF gene in extreme mastitis susceptibility cows (126 Holstein and Holstein crossbred) was performed.. Eight polymorphisms were found in promotor region, four of them were novel variations. Two were important by frequency among extreme groups, but a polymorphism in - 421 A/T position was significantly (P = 0.0188) associated to mastitis susceptibility.. Results support the key role of regulatory region of LTF gene. Some candidate genes are proposed in association with mastitis traits and implications are discussed.

Topics: Animals; Cattle; Female; Genotype; Humans; Lactoferrin; Mastitis, Bovine; Milk; Polymorphism, Genetic; Polymorphism, Single Nucleotide

The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and 5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group, pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188. Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi. In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of IL-1β induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoculated with strain 5011 (P) showed similar IL-1β levels to those found in control quarters. In quarters inoculated with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher total IgG and IgG

Topics: Animals; Cattle; Cell Count; Female; Genotype; Immunity; Immunoglobulin G; Interleukin-6; Lactation; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Nitric Oxide; Saline Solution; Staphylococcal Infections; Staphylococcus aureus

Variations in the levels of acute phase proteins and lactoferrin in serum and milk for diagnosis of subclinical mastitis in dairy cows are described in this research paper. Milking animals from two organized dairy farms in Kerala, India, were screened by California Mastitis Test (CMT), Electrical Conductivity test (EC) and Somatic Cell Count (SCC) test to identify animals affected with sub clinical mastitis (SCM). The concentrations of acute phase proteins (APP) Haptoglobin (Hp), C- reactive protein (CRP), Albumin, Lactoferrin (Lf) and α- 1 acid glycoprotein (AGP) in milk and Hp, Albumin, Serum Amyloid A (SAA) and CRP in the serum of 40 normal cows and 40 cows affected with sub clinical mastitis were assessed. Solid phase ELISA was employed for assessment of all parameters except the albumin levels, for which spectrophotometry was used. The values of Hp in milk; and SAA, AGP and Lf in serum, were significantly elevated in the group with sub clinical mastitis. Such variations were found to be independent of the specific bacterial organism causing the disease. These results show that significant variations exist in the levels of acute phase proteins Hp, AGP and Lf in milk, and SAA in serum of animals affected with subclinical bovine mastitis that are not affected by specific bacterial etiology.

Topics: Acute-Phase Proteins; Animals; Biomarkers; C-Reactive Protein; Cattle; Cell Count; Female; Haptoglobins; India; Lactoferrin; Mass Screening; Mastitis, Bovine; Milk; Serum Amyloid A Protein

Lactoferricin (Lfcin) is an antimicrobial activity center of lactoferrin, produced by hydrolysis from the N-terminal of lactoferrin. It was hypothesized that the intramolecular disulfide bond in Lfcin could affect its antibacterial function through influencing its molecular structure. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with an intramolecular disulfate bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. The antibacterial activity of the peptides against Trueperella pyogenes, separated from cow milk with mastitis, were determined.. The secondary structure of bLfcin DB showed more β-turn and less random coil than the other peptides in H. The intramolecular disulfide bond could change the molecular structure of bLfcin under alternative ionic strengths and hydrophobic effects, and the formation of the disulfide bond is beneficial to executing the antibacterial function of bLfcin.

Topics: Actinomycetaceae; Animals; Anti-Bacterial Agents; Cattle; Circular Dichroism; Disulfides; Escherichia coli; Female; Lactoferrin; Mastitis, Bovine; Milk; Molecular Structure; Protein Structure, Secondary

Treatment of bovine mastitis with intramammary antibiotics is common, yet several concerns exist including failed efficacy for individual hosts or pathogens and the inability of approved drugs to revert mastitis-induced tissue damage to healthy tissue capable of returning to full milk production. These issues, in addition to aspects of public health such as accidental antibiotic residues in saleable milk and the potential for antimicrobial resistance, support the need to find alternative therapies for this costly disease. This study shows that the secretome, or collective factors, produced by mammosphere-derived cells (MDC) promotes angiogenesis, epithelial cell migration, and contains proteins associated with immunity and defense; all of which are necessary for healing damaged mammary gland tissue. Furthermore, we found that the MDC secretome remains effective after freezing and thawing, enhancing its therapeutic potential. Our results provide a foundation for further characterization of the individual secreted factors and the rationale for using the MDC secretome as a complementary treatment for bovine mastitis.

Topics: Angiopoietin-1; Animals; Bacterial Toxins; Cattle; Cell Movement; Chromatography, High Pressure Liquid; Epithelial Cells; Female; Lactoferrin; Lipopolysaccharides; Mammary Glands, Animal; Mass Spectrometry; Mastitis, Bovine; Milk; Neovascularization, Physiologic; Transforming Growth Factor beta

During the resolution of inflammation, macrophages engulf apoptotic polymorphonuclear cells (PMN) and can accumulate large numbers of their corpses. Here, we report that resolution phase macrophages acquire the neutrophil-derived glycoprotein lactoferrin (Lf) and fragments thereof

Topics: Animals; Apoptosis; Cattle; Cells, Cultured; Cytokines; Disease Models, Animal; Extracellular Traps; Female; Humans; Inflammation; Lactoferrin; Macrophages; Male; Mastitis, Bovine; Mice; Mice, Inbred C57BL; Neutrophils; Peptide Fragments; Peritonitis; Phagocytosis

Streptococcus dysgalactiae ssp. dysgalactiae is an important causative agent of bovine mastitis worldwide. Lactoferrin is an innate immune protein that is associated with many functions including immunomodulatory, antiproliferative, and antimicrobial properties. This study aimed to investigate the interactions between lactoferrin and a clinical bovine mastitis isolate, Strep. dysgalactiae ssp. dysgalactiae DPC5345. Initially a deliberate in vivo bovine intramammary challenge was performed with Strep. dysgalactiae DPC5345. Results demonstrated a significant difference in lactoferrin mRNA levels in milk cells between the control and infused quarters 7h postinfusion. Milk lactoferrin levels in the Strep. dysgalactiae DPC5345 infused quarters were significantly increased compared with control quarters at 48h postinfusion. In vitro studies demonstrated that lactoferrin had a bacteriostatic effect on the growth of Strep. dysgalactiae DPC5345 and significantly decreased the ability of the bacteria to internalize into HC-11 mammary epithelial cells. Confocal microscopy images of HC-11 cells exposed to Strep. dysgalactiae and lactoferrin further supported this effect by demonstrating reduced invasion of bacteria to HC-11 cells. The combined data suggest that a bovine immune response to Strep. dysgalactiae infection includes a significant increase in lactoferrin expression in vivo, and based on in vitro data, lactoferrin limits mammary cell invasion of this pathogen by binding to the bacteria and preventing its adherence.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cattle Diseases; Epithelial Cells; Female; Immunity, Innate; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; RNA, Messenger; Streptococcal Infections; Streptococcus

The objectives of the present study were to investigate the change in the number of viable pathogens during preservation of milk obtained from cows with subclinical mastitis and the association between the decreasing ratio of viable bacteria during preservation and the somatic cell count (SCC) and the values of lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO). After preservation of milk at room temperature for 0, 0.5, 1, 2, 3, 4 and 5 hr, the bacterial colonies in the milk were counted to determine the number of colony forming units (CFUs). Fresh skim milk was used to determine the values of LAP, LPO and LF. Bacteria were not detected in 19.4% of milk samples, and this percentage increased up to 30% after 5 hr of preservation. The number of Staphylococcus aureus and Streptococcus uberis in milk did not change significantly during the 5-hr incubation, whereas significant decreases were observed in the number of coliforms, coagulase-negative staphylococci, yeasts and Corynebacterium bovis. High SCC significantly decreased CFUs of S. aureus and yeast after preservation of milk for 4 to 5 hr. High LF concentration in milk was associated with decrease in CFU of S. aureus during 4-hr preservation. These results suggest that the viable counts of some pathogens in milk decreased during preservation at room temperature after collection, which may be attributed to the leukocytes and antimicrobial components present in milk.

Topics: Animals; Asymptomatic Infections; Bacterial Load; beta-Defensins; Cattle; Female; Food Preservation; Lactoferrin; Lactoperoxidase; Mastitis, Bovine; Milk

The objective of this study was to identify and characterize potential biomarkers for disease resistance in bovine milk that can be used to indicate dairy cows at risk to develop future health problems. We selected high- and low-resistant cows i.e. cows that were less or more prone to develop diseases according to farmers' experience and notifications in the disease registration data. The protein composition of milk serum samples of these high- and low-resistant cows were compared using NanoLC-MS/MS. In total 78 proteins were identified and quantified of which 13 were significantly more abundant in low-resistant cows than high-resistant cows. Quantification of one of these proteins, lactoferrin (LF), by ELISA in a new and much larger set of full fat milk samples confirmed higher LF levels in low- versus high-resistant cows. These high- and low-resistant cows were selected based on comprehensive disease registration and milk recording data, and absence of disease for at least 4 weeks. Relating the experienced diseases to LF levels in milk showed that lameness was associated with higher LF levels in milk. Analysis of the prognostic value of LF showed that low-resistant cows with higher LF levels in milk had a higher risk of being culled within one year after testing than high-resistant cows. In conclusion, LF in milk are higher in low-resistant cows, are associated with lameness and may be a prognostic marker for risk of premature culling.

Topics: Animals; Biomarkers; Cattle; Cattle Diseases; Disease Resistance; Female; Lactoferrin; Lameness, Animal; Mastitis, Bovine; Milk; Prognosis; Proteomics; Tandem Mass Spectrometry

The aim of this study was to analyze an effect of udder health status, somatic cell count (SCC), stage and number of lactations, and different seasons on the concentration of lactoferrin (LF) and immunoglobulin G (IgG) in quarter milk samples (n=120) from crossbreed (Lithuanian Black-and-White & Holstein) dairy cows. Quarter health status was based on SCC and microbiological analysis. The highest mean value of LF and IgG were observed in quarters with subclinical mastitis 0.1 ± 0.02 mg/ml and 0.41 ± 0.06 mg/ml, respectively. Grouping the data according to SCC revealed increased LF (0.07 ± 0.01 mg/ml as against 0.06 ± 0.01 mg/ml) and IgG values (0.27 ± 0.05 mg/ml as against 0.23 ± 0.02 mg/ml) in DQ (SCC from 201,000 ≥ 401,000 cells/ml) compared to HQ (SCC up to 200,000 cells/ml). The milk LF and IgG levels were effected by stage of lactation (p<0.01 and p<0.05, respectively) and season of the year (p<0.001 and p<0.001, respectively). Nevertheless, SCC and subsequent lactation (p>0.05) had no effect on these immunity components.

Topics: Animals; Cattle; Female; Immunoglobulin G; Lactation; Lactoferrin; Mastitis, Bovine; Milk; Seasons

The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day -6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days -6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 (<5 × 10(6) cell/mL) were significantly higher and lower than those in cows with a higher SCC, respectively. These results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factor in milk of cows treated for clinical mastitis.

Topics: Animals; Anti-Bacterial Agents; beta-Defensins; Cattle; Cefazolin; Immunity, Innate; Lactoferrin; Lactoperoxidase; Mastitis, Bovine; Milk

Lactoferrin (Lf) gene promoter was screened for the presence of single nucleotide polymphism in indigenous and crossbred cattle from North India and to evaluate its association with Mastitis. Study revealed the presence of genetic variation in regulatory region of bovine Lactoferrin gene using PCR-RFLP technique. Three genotypes namely GG, GH and HH were identified. A single nucleotide change, from guanine to adenine at 25th position was found to be significantly associated (p<0.05) with clinical mastitis in indigenous Sahiwal and crossbred Karan Fries cattle maintained at organised herd of National Dairy Research Institute, Karnal. A non-significant association was observed between subclinical mastitis, somatic cell score (SCS), and GG genotype in Karan Fries cattle, however, a lower SCS was observed in animals having GG genotype. Overall a lower incidence of clinical mastitis was recorded in those animals having GG genotype of Lf in Sahiwal and Karan Fries (KF) cattle. The SNP identified in the promoter region may effect expression lactoferrin protein, which may lead to different levels of antibacterial and anti-inflammatory activity of Lf gene. Results from this study indicated the probable role played by Lactoferrin promoter to serve as candidate gene for mastitis susceptibility among indigenous and crossbred milch cattle.

Topics: Animals; Base Sequence; Cattle; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Genotype; Lactoferrin; Mastitis, Bovine; Promoter Regions, Genetic

Under the traditional grazing system on the Qinghai-Tibetan Plateau, the amount of milk in domesticated yak (Bos grunniens) with clinical mastitis decreases and the milk composition is altered. To understand the mechanisms of mammary gland secreted milk and disease infection, changes in the protein composition of milk during clinical mastitis were investigated using a proteomic approach. Milk whey from yak with clinical mastitis was compared to whey from healthy animals with two-dimensional gel electrophoresis using a mass spectrometer. Thirteen protein spots were identified to be four differentially expressed proteins. Increases in the concentrations of proteins of blood serum origin, including lactoferrin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins, casocidin-I, a-lactalbumin, and b-lactoglobulin, were downregulated in mastitic whey. These results indicated significant differences in protein expression between healthy yaks and those with clinical mastitis, and they may provide valuable information for finding new regulation markers and potential protein targets for the treatment of mastitis.

Topics: Amino Acid Sequence; Animals; Caseins; Cattle; Electrophoresis, Gel, Two-Dimensional; Female; Lactalbumin; Lactoferrin; Lactoglobulins; Mass Spectrometry; Mastitis, Bovine; Milk; Milk Proteins; Molecular Sequence Data; Peptide Fragments; Proteome; Proteomics; Whey Proteins

This study analyzed the association between single nucleotide polymorphism (A/C) in position -28 located in the TATA box of LTF gene and the lactoferrin concentration in bovine milk secreted by healthy and infected udders. Out of 241, 69 cows were selected into the experimental group and were divided into 3 groups according to mean value of somatic cell count (SCC): I < 180,000 cells/mL, II: 180,000-350,000 cells/mL and III > 350,000 cells/mL. In each SCC group, three LTF genotypes: AA, AC and CC were identified by PCR-SSCP method. A total of 604 milk samples were collected monthly and lactoferrin concentration was measured by ELISA. The 1-way ANOVA within SCC groups was performed to estimate association of -28 A/C genotypes with mean lactoferrin concentration per lactation. In the group of healthy cows (< 180,000 cells/mL) LTF concentration in milk cows with the AA genotype (107.58 ± 17.92 μg/mL) was significantly higher than in homozygotes CC (52.09 ± 19.01 μg/mL). Unexpectedly, in cows with elevated SCC (> 350,000 cells/mL) we observed a significant opposite relationship (207.21 ± 28.50 in CC vs 115.0 ± 28.6 μg/mL in AA). We hypothesized that a promoter with allele C, which cannot be recognized as a TATA sequence is becoming more accessible for other transcription factors, which may induce alternative LTF gene expression. We assume that our results demonstrate a very interesting effect of differential gene expression depending on polymorphism in a key regulatory motif (TATA box) and also on the health status of mammary tissues.

Topics: Animals; Cattle; Female; Genotype; Lactoferrin; Mastitis, Bovine; Milk; Polymorphism, Genetic; Promoter Regions, Genetic

One major problem in dairy cattle husbandry is the prevalence of udder infections. In today's breeding programmes, top priority is being given to making animal evaluation more cost-effective and reliable and less time-consuming. We proposed tumor necrosis factor α (TNF-α), lactoferrin (LTF) and macrophage-expressed lysozyme (mLYZ) genes as potential DNA markers in the improvement of immunity to mastitis.This study included 588 Polish Holstein-Friesian cows kept on one farm located in the north-western region of Poland. All clinical cases of mastitis in the herd under study were recorded by a qualified veterinarian employed by the farm. The following indicators were applied to determine udder immunity to mastitis in the cows under study: morbidity rate (MR), duration of mastitis (DM) and extent of mastitis (EM). TNF-α, mLYZ and LTF genotypes were identified by real-time PCR method, using SimpleProbe technology. Due to the very low frequency of mLYZ allele T, the gene was excluded from further analysis.A statistical analysis of associations between TNF-α and LTF genes and immunity to mastitis were performed using three models: 1) a parity-averaged model including only additive effects of the genes; 2) a parity-averaged model including both additive and epistatic effects of the genes; and 3) a parity-specific model including only additive effects of the genes.. With the first and second models it was revealed that the genes effects on the applied indicators of immunity to mastitis were non-significant whereas with the third one the effects were found to be statistically significant. Particularly noteworthy was the finding that the effects of TNF-α and LTF varied depending on age (parity). The alleles which were linked to high immunity to mastitis in lower parities appeared to be less favourable in higher parities.. These interactions might be related to inflamm-ageing, that is an increased susceptibility to infection due to immune system deregulation that progresses with age. Such pattern of interactions makes it impossible to use the genes in question in marker-assisted selection aimed at reducing heritable susceptibility to mastitis. This is because the immune mechanisms behind resistance to infections proved to be too complex.

Topics: Animals; Cattle; Female; Gene Frequency; Genetic Markers; Genetic Predisposition to Disease; Genotype; Lactoferrin; Mastitis, Bovine; Muramidase; Parity; Polymorphism, Genetic; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

The objective of the current study was to analyze the variations in lactoferrin (LF) concentrations in primiparous cows with intramammary infection and to study how the lactation stage affects these variations. In addition, we aimed to study the potential of the LF concentration in early lactation as a predictive factor for future infections. To accomplish this goal, a longitudinal analysis was performed for 96 primiparous cows. Milk samples were collected each month from individual quarters, and the LF concentration was determined for each sample. Criteria that included both somatic cell count (SCC) and a microbiological analysis were used to assess the health status of the quarters. Of the diseased quarters (SCC >200,000 or positive for pathogen isolation, or both), 62% corresponded to nonspecific mastitis (SCC >200,000 but microbiologically negative) and 25% corresponded to the category "presence of bacterial growth" (SCC <200,000 but microbiologically positive). Diseased quarters showed increased concentrations of LF compared with healthy quarters. However, this increase was greater during the first days of lactation compared with later periods. Kaplan-Meier analysis of time free of infection demonstrated that quarters with LF concentrations at early lactation (3-10d in milk) greater than 0.1mg/mL are more likely to become infected during the following lactation compared with quarters with lower LF concentrations in early lactation. The results support that LF plays a relevant role in combating intramammary infection, particularly during the first days of lactation. In addition, we present evidence of the potential use of LF as a predictive marker of future infections in the individual quarters of dairy heifers.

Topics: Animals; Cattle; Cell Count; Female; Lactation; Lactoferrin; Mastitis, Bovine; Milk

Mastitis remains a major cattle disease with great global economic implications. Various approaches are currently employed in attempts to improve understanding of mastitis resistance and develop phenotypic markers for use in breeding programs (e.g., somatic cell score), including QTL discovery, wide-genome association studies, and identification of candidate genes related to immune function. This study evaluated three single nucleotide polymorphisms contained in Toll-like receptor 4 (TLR4) and lactoferrin (LF) genes associated with mastitis traits: TLR4 P-226, TLR4 2021, and LF P-28. Genotyping was performed by restriction fragment length polymorphism-polymerase chain reaction (PCR) and high-resolution melting quantitative PCR from genomic DNA of four dairy cattle breeds (Holstein, Jersey, Montbeliarde, and Overo Colorado) previously classified as healthy, with clinical or with subclinical mastitis. The high-resolution melting quantitative PCR allowed genotyping of each locus and resulted in allele frequencies indicating that all loci were in Hardy-Weinberg equilibrium. The TT genotype of TLR4 2021 was significantly associated with the healthy condition, but no associations with somatic cell score were evident. Further studies are therefore necessary in order to confirm the results of this investigation.

Topics: Animals; Animals, Inbred Strains; Cattle; Female; Genetic Association Studies; Genetic Linkage; Lactoferrin; Mastitis, Bovine; Polymorphism, Single Nucleotide; Toll-Like Receptor 4

Pheromonicin-SA (Ph-SA) is a newly developed, engineered multidomain peptide that has a bactericidal effect against Staphylococcus aureus. The objective of this study was to characterize innate immune responses by Staph. aureus-stimulated bovine mammary epithelial cells (BMEC) following treatment with Ph-SA. Primary BMEC from one lactating Holstein cow were isolated and exposed to Staph. aureus for 2 h, and then treated with rifampicin or Ph-SA. Total RNA was isolated from BMEC at 0, 2, 6, 12, and 24 h postinfection, and the mRNA expression of selected genes, including toll-like receptor (TLR)2 and TLR4, IL-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α), and lactoferrin, was quantified by real-time PCR. In the rifampicin group, increases in the expression of mRNA for TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection and in the expression of mRNA for TLR2 but not for TLR4 at 12 h postinfection. In the Ph-SA group, increases in the mRNA expression of TLR2, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection, and an increase in TLR4 mRNA expression was observed at 24 h postinfection. At 24 h postinfection, the mRNA expression of TLR4, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin was higher in the Ph-SA group than in the rifampicin group. In conclusion, Ph-SA might promote the expression of mRNA for TLR2, TLR4, the pro-inflammatory cytokines IL-1, IL-6, and TNF-α, the chemotactic factor IL-8, and lactoferrin in Staph. aureus-infected BMEC. Moreover, Ph-SA may be of value as an antibiotic in promoting innate immune responses by Staph. aureus-infected bovine mammary epithelial cells.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cytokines; Epithelium; Female; Interleukin-1beta; Interleukin-6; Interleukin-8; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Recombinant Fusion Proteins; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Lactoferrin (LTF) is a milk glycoprotein favorably associated with the immune system of dairy cows. Somatic cell count is often used as an indicator of mastitis in dairy cows, but knowledge on the milk LTF content could aid in mastitis detection. An inexpensive, rapid and robust method to predict milk LTF is required. The aim of this study was to develop an equation to quantify the LTF content in bovine milk using mid-infrared (MIR) spectrometry. LTF was quantified by enzyme-linked immunosorbent assay (ELISA), and all milk samples were analyzed by MIR. After discarding samples with a coefficient of variation between 2 ELISA measurements of more than 5% and the spectral outliers, the calibration set consisted of 2499 samples from Belgium (n = 110), Ireland (n = 1658) and Scotland (n = 731). Six statistical methods were evaluated to develop the LTF equation. The best method yielded a cross-validation coefficient of determination for LTF of 0.71 and a cross-validation standard error of 50.55 mg/l of milk. An external validation was undertaken using an additional dataset containing 274 Walloon samples. The validation coefficient of determination was 0.60. To assess the usefulness of the MIR predicted LTF, four logistic regressions using somatic cell score (SCS) and MIR LTF were developed to predict the presence of mastitis. The dataset used to build the logistic regressions consisted of 275 mastitis records and 13 507 MIR data collected in 18 Walloon herds. The LTF and the interaction SCS × LTF effects were significant (P < 0.001 and P = 0.02, respectively). When only the predicted LTF was included in the model, the prediction of the presence of mastitis was not accurate despite a moderate correlation between SCS and LTF (r = 0.54). The specificity and the sensitivity of models were assessed using Walloon data (i.e. internal validation) and data collected from a research herd at the University of Wisconsin - Madison (i.e. 5886 Wisconsin MIR records related to 93 mastistis events - external validation). Model specificity was better when LTF was included in the regression along with SCS when compared with SCS alone. Correct classification of non-mastitis records was 95.44% and 92.05% from Wisconsin and Walloon data, respectively. The same conclusion was formulated from the Hosmer and Lemeshow test. In conclusion, this study confirms the possibility to quantify an LTF indicator from milk MIR spectra. It suggests the usefulness of this indicator associated to SCS

Topics: Animals; Calibration; Cattle; Enzyme-Linked Immunosorbent Assay; Female; Lactoferrin; Mastitis, Bovine; Milk; Reproducibility of Results; Spectrophotometry, Infrared

Streptococcus uberis is the most common environmental mastitis pathogen causing udder inflammations of different severities in dairy cows. The aim of the study was to investigate if the different clinical outcome of mastitis induced by different strains of S. uberis can be reflected in the mammary immune response. Mammary epithelial cells and somatic milk cells were treated with heat inactivated and living S. uberis of strain A and strain B in vitro. Strain A was repeatedly isolated from a chronically infected quarter during 8 months, and persisted in the quarter despite antibiotic treatment. Strain B caused an acute clinical mastitis and was not further isolated after a single antibiotic treatment. Treatment with Strain B induced a more pronounced increase of mRNA-expression of various immune factors (interleukin-8, interleukin-1beta, RANTES, and lactoferrin) in mammary epithelial cells than strain A. In contrast to mammary epithelial cells the response of removed somatic milk cells showed no differences between the stimulation with two S. uberis strains. Tumor necrosis factor-alpha mRNA expression was not differently induced by the two strains. In conclusion, the characteristics of different severities of mastitis that are induced by different S. uberis strains in vivo can also be reflected at the level of the immune response of the mammary gland in vitro.

Topics: Animals; Cattle; Cells, Cultured; Chemokine CCL5; Cytokines; Epithelial Cells; Female; Gene Expression Regulation, Bacterial; Interleukin-1beta; Interleukin-8; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; RNA, Messenger; Severity of Illness Index; Streptococcal Infections; Streptococcus; Tumor Necrosis Factor-alpha

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

Topics: Alternative Splicing; Animals; Cattle; Exons; Female; Gene Expression; Lactoferrin; Liver; Mammary Glands, Animal; Mastitis, Bovine; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Staphylococcal Infections; Staphylococcus aureus

Lactoferrin (Lf) is naturally produced by the mammary gland, having biological functions of antibacterial and anti-inflammatory activities. To investigate whether the Lf gene is associated with mastitis in dairy cattle, a DNA sequencing approach was used to identify single nucleotide polymorphisms (SNPs) in the gene. Three previously reported SNPs in the 5' flanking region and one novel SNP in exon1 of Lf gene were identified. A total of 353 individuals from Holstein cattle populations were genotyped for their SNPs using Created Restriction Site PCR (CRS-PCR) and PCR-RFLP methods. Twenty-two and nineteen combinations of three SNPs (g.3440T>G, g.3879_3880insG, and g.4432T>C) and another three SNPs (g.3429G>A, g.3440T>G, g.3879_3880insG) were observed, respectively. The result of haplotype analysis of four SNPs showed that fourteen different haplotypes were identified. Two major haplotypes (GECB and GECA) occurred with a frequency of 22.5 and 18.5% in the study population, respectively. Statistical analyses revealed no significant association between one single SNP of Lf gene and SCS, whereas significant associations between their combined genotypes of three SNPs, haplotype and SCS. Combined genotype EFCDBB and GGEFDD with the lowest SCS were favorable for the mastitis resistance. They may be used as a possible candidate for marker-assisted selection in dairy cattle breeding program.

Topics: 5' Flanking Region; Animals; Base Sequence; Cattle; China; Exons; Female; Gene Frequency; Genetic Predisposition to Disease; Haplotypes; Heterozygote; Lactoferrin; Mastitis, Bovine; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Sequence Analysis, DNA

The dry period plays an important role in maintenance of udder health. Cows are most susceptible to intramammary infections (IMI) after dry-off and near parturition and drying-off procedures may affect the likelihood of IMI at calving. The objective of this study was to evaluate the association of milk yield and infection status at dry-off with the likelihood of IMI at calving by examining different drying-off methods. Cows (n=112) at the Ohio State University Waterman Dairy Teaching and Research Herd were randomly assigned to either an intermittent or a standard, twice-daily milking group 1 week prior to dry-off. All quarters of all cows in the herd were treated with an antibiotic dry-cow product after the last milking. Milk samples were collected 1 week prior to dry-off (pre-dry), on the day of dry-off, and within 3 d of parturition to determine infection status of the quarters. Association between IMI at calving and cumulative milk yield for the final week of lactation and drying-off method was examined using generalized estimation equations with logic link, accounting for potential confounders, such as pre-dry and dry-off infection status, and for the correlated data structure due to quarters clustered within cows. Intermittent milking significantly reduced milk yield at the end of lactation. Increasing cumulative milk yield during the last week of lactation was significantly associated with a greater probability of IMI at calving for quarters that were uninfected prior to dry-off: uninfected quarters of cows producing more than 115 kg during the last week of lactation were 7.1-times more likely to be infected at calving (P=0.0081) than uninfected quarters of cows producing less than 75 kg. Even though the overall cure rate over the dry period was relatively high at 84%, the odds of a quarter being infected at calving was 7.6- and 3.3-times higher if it was infected at dry-off with major pathogens (P<0.0001) or minor pathogens (P=0.028), respectively, compared with an uninfected quarter at dry-off. The results suggest that decreasing milk yield prior to dry-off may serve as an effective means to maintain good udder health in a herd.

Topics: Animals; Cattle; Dairying; Female; Lactation; Lactoferrin; Logistic Models; Mammary Glands, Animal; Mastitis, Bovine; Milk; Parturition; Pregnancy; Seasons; Time Factors

Six transgenic cows producing recombinant human lactoferrin (rhLf) in their milk and five normal cows at the same lactation stage were experimentally infected with Staphylococcus chromogenes to study the effect of a high concentration of lactoferrin in milk. Coagulase-negative staphylococci such as S. chromogenes have become very common as agents causing mild or subclinical mastitis. All transgenic cows became infected but showed no clinical signs, unlike the control cows, which developed mild clinical mastitis. Transgenic cows eliminated bacteria faster from the quarters than did the controls. Local clinical signs were milder, and the inflammatory reaction assessed by NAGase activity in the milk and by the concentration of milk amyloid A was lower in the transgenic cows. The mild response probably reflected the rapid elimination of bacteria. The milk concentration of rhLf remained constant throughout the study period, but the total concentration of bovine lactoferrin in the milk peaked in both groups at 46h post-challenge. Three cows, all in the control group, exhibited systemic acute phase response as increased concentrations of serum amyloid A in the blood circulation. Transgenic cows with a high concentration of human lactoferrin in their milk seemed to be protected from clinical disease and from prolonged inflammatory reaction, but not from experimental intramammary infection induced by S. chromogenes.

Topics: Acetylglucosaminidase; Animals; Animals, Genetically Modified; Cattle; Cell Count; Female; Humans; Lactation; Lactoferrin; Linear Models; Mastitis, Bovine; Milk; Serum Amyloid A Protein; Staphylococcal Infections; Staphylococcus

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Topics: Animals; Cattle; Citrates; Female; Lactation; Lactoferrin; Lipopolysaccharides; Mastitis, Bovine; Milk; Time Factors

Bovine lactoferrin (LF) is a multifunctional glycoprotein found in milk, which acts mainly as a defense factor in the mammary gland. Polymorphism has been found in the bovine LF gene. However, there is no report on genetic polymorphism of LF gene and its associations with mastitis in dairy cattle. In this study, the promoter fragment of LF gene containing -926(G/A), -915(T/G), -478(/G), and +72(T/C) mutations were genotyped by the PCR-RFLP and CRS-PCR method. Two hundred and sixty-eight Chinese Holstein cows were screened. Least square linear model (LSM) analysis was applied to evaluate the associations of LF gene with somatic cell score (SCS). The results indicated that the SCS was significantly affected by -478(/G) and +72(T/C), but not by the other two loci (P >0.05). The SCS of cow with genotype AB in +72(T/C) position was significantly lower than that of genotype AA (P<0.01) or AB (P<0.05). In position -478(/G), the cow with genotype CC showed significantly lower SCS in contrast to cow with genotype CD and DD (P < 0.01). In conclusion, genotype AB in position +72(T/C) and genotype CC in position -478(/G) of LF gene were advantageous genotype, which can be used as candidate markers for mastitis resistance selection in dairy cattle.

Topics: 5' Flanking Region; Animals; Cattle; Female; Genetic Predisposition to Disease; Genotype; Lactoferrin; Mastitis, Bovine; Polymerase Chain Reaction; Polymorphism, Genetic

Lactoferrin (LF) concentrations in the milk with different levels of the somatic cell count score were examined using an ELISA to determine whether milk LF concentration is influenced by parity of the cow, stage of lactation, and the somatic cell count. The study animals were 198 Chinese Holstein cows randomly chosen from more than 1,600 cows in 4 dairy farms in the Beijing area. The cows had shown no sign of mastitis for 2 mo. Daily milk production was recorded, and milk samples were taken from individual cow samples. The LF concentration varied between 31.78 and 485.63 microg/mL in milk from normal animals. Lactoferrin was significantly associated with stage of lactation (r = 0.557) and daily milk production (r = -0.472). Nevertheless, there was no significant relationship with parity. Moreover, milk LF concentration tended to be correlated with the somatic cell count score (r = 0.375). This finding suggests that milk LF may be helpful as an indicator for intramammary infection in dairy cows.

Topics: Animals; Cattle; Female; Lactation; Lactoferrin; Mastitis, Bovine; Milk; Parity; Pregnancy; Time Factors

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.

Topics: Animals; Cattle; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Species Specificity; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus

Pathogens invading the mammary gland cause a complex signaling network that activates the early immune defense and leads to an outcome of inflammation symptoms. To examine the importance of mammary epithelial cells in these regulations and interactions resulting in a pathogen-related course of mastitis, we characterized the mRNA expression profile of key molecules of the innate immune system by quantitative real-time PCR. Mammary gland epithelial cells isolated on d 42 of lactation from 28 first-lactation Holstein dairy cows were cultured separately under standardized conditions and treated for 1, 6, and 24 h with heat-inactivated gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Both pathogens increased mRNA expression patterns of proteins involved in pathogen recognition such as Toll-like receptors and nuclear factor-kappa B, whereas gram-negatives acted as a stronger stimulus. Furthermore, this could be confirmed by the expression profile of the proinflammatory cytokines tumor necrosis factor alpha, IL-1 beta, IL-6, and chemokines such as IL-8 and RANTES (regulated upon activation, normal T-cell expressed and secreted). Remarkably, at a low level of mRNA expression after 1 h of treatment these cytokines and chemokines were expressed at a significantly higher level in Staphyloccocus aureus than in Escherichia coli affected cells. Lactoferrin showed a deviating expression pattern to pathogen stimulation (i.e., at the 1-h measuring point Escherichia coli induced a higher mRNA expression, whereas the highest level was reached after 24 h of stimulation with Staphylococcus aureus). Complement factor 3 was the only measured factor that responded equally to both microorganisms. Our data emphasize the role of mammary epithelial cells in the immune defense of the udder and confirm their contribution to pathogen-related different courses of mastitis.

Topics: Animals; Cattle; Cytokines; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Antibacterial activity of bovine lactoferrin hydrolysates (LFH) on microorganisms isolated from bovine mastitis, and superoxide (O(2)(-)) production of bovine neutrophils were evaluated. Antibacterial effects of LFH were measured in vitro against Staphylococcus aureus, coagulase-negative staphylococci, Streptococci, Enterococci, Escherichia coli, Klebsiella pneumoniae, yeast-like fungi and Prototheca zopfii isolated from clinical cases of bovine mastitis. To compare susceptibilities against LFH, minimal inhibitory concentration (MIC) values were determined by a micro-plate assay method. Most organisms were sensitive to LFH. Prototheca zopfii was highly sensitive to LFH; the growth of the microorganism was inhibited completely even at 1 mug/ml. Staphylococcus aureus and Escherichia coli were resistant to LFH. The production of O(2)(-) by bovine neutrophils was used to evaluate the effect of LFH administration on functional activity. Increase in O(2)(-) production by bovine neutrophils occurred upon addition of LFH to neutrophils. These results demonstrate that LFH possesses antibacterial activity against pathogens that cause mastitis and activates neutrophil superoxide production.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Cattle; Dose-Response Relationship, Drug; Female; Fungi; Lactoferrin; Mastitis, Bovine; Microbial Sensitivity Tests; Milk; Neutrophils; Protein Hydrolysates; Superoxides

The efficacy of intramammary (IM) treatments containing penicillin G (PG) alone or a combination of PG and bovine lactoferrin (bLF) was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of Staphylococcus aureus highly resistant to beta-lactam antibiotics. First, we confirmed that this strain could cause mastitis and infection could not be cured with PG alone. In a second trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of Staph. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 IM treatments as follows: 1) citrate buffer, 2) 100,000 IU of PG, (3) 1 g of bLF, or 4) 1 g of bLF + 100,000 IU of PG. Treatments were repeated twice a day for 5 d. A third trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month before dry-off, 20 gravid cows regrouped by dates of calving were infected in their 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to 1 of 2 treatments as follows: 1) 100,000 IU of PG or 2) 250 mg of bLF + 100,000 IU of PG. Treatments were administered IM twice a day for 7 d. For all trials, milk samples were taken to monitor bacterial concentration and somatic cell count. Bacteriological cure rate was determined using milk samples taken 3 and 4 wk after initiation of treatments. For the second trial, cure rate was null for control quarters, 11.1% for bLF, 9.1% for PG, and 45.5% for the bLF + PG combination. For cows infected in their previous lactation, cure rate was higher for the bLF + PG combination (33.3%) compared with PG alone (12.5%). In conclusion, bLF added to PG is an effective combination (i.e., 3- to 5-times higher cure rate) for the treatment of stable Staph. aureu infections highly resistant to beta-lactam antibiotics.

Topics: Animals; Anti-Bacterial Agents; beta-Lactam Resistance; Cattle; Cell Count; Drug Synergism; Drug Therapy, Combination; Female; Lactoferrin; Mastitis, Bovine; Milk; Penicillins; Random Allocation; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

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.

Topics: Animals; Cattle; Colostrum; Concanavalin A; Female; Immunoglobulin G; Lactoferrin; Mastitis, Bovine

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.

Topics: Amino Acid Sequence; Animals; Cattle; Concanavalin A; Epithelial Cells; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Molecular Sequence Data; Pancreatic Elastase; Staphylococcal Infections

This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.

Topics: Animals; Animals, Genetically Modified; Carrier Proteins; Cattle; Escherichia coli; Escherichia coli Infections; Female; Humans; Lactoferrin; Mastitis, Bovine; Recombinant Proteins

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.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cattle; Cattle Diseases; Chromatography, Affinity; Concanavalin A; Cytokines; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Female; Immunoelectrophoresis, Two-Dimensional; Iron; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Molecular Sequence Data; Oxygen; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, Protein; Staphylococcus aureus; T-Lymphocytes; Tumor Necrosis Factor-alpha

Leucocytes (WBC) are recruited from peripheral blood into milk as part of the inflammatory response, mediated through cytokines or interleukins (IL) synthesized by mammary tissue and the milk somatic cells (SC). The inflammatory response is related to the concentration of SC and the cytokines produced. To investigate and to compare the kinetics of cytokine production in SC and WBC during inflammation, cell culture models were established, where SC and WBC were cultured in parallel (n = 3). In addition, macrophages or monocytes were isolated from milk and blood with antibody-coated magnetic beads and cultivated separately. Isolated cells were pure, unaltered and viable. Cultures were activated with 10 microg/ml lipopolysaccharide (LPS). After 0, 1, 2, 3, 4 and 8 h cells were harvested for RNA isolation. Cytokine [tumour necrosis factor alpha (TNFalpha), IL-1beta, IL-6] mRNA expression responses and transcriptional activity of CD14 and lactoferrin (LF) were quantified via a one-step real-time RT-PCR. Significant cytokine mRNA increases were found in all four cell culture types and genes, with peaks after 1 and 2 h (TNFalpha > IL-6 > IL-1beta). In WBC or monocytes higher LPS responses and longer persistence could be found than in corresponding milk cells (IL-1beta > IL-6 > TNFalpha). SC and macrophages are less responsive to LPS stimulation than WBC or monocytes. The strength of the immune response in the blood system is much more prominent than in the mammary gland. This may be ascribed to the role of CD14 on the cytokine production of the investigated cells, or may be caused by the blood-to-milk diapedesis. The constitutive transcription of CD14 mRNA in WBC and monocytes was found to be 6 to 15 times higher than in adequate milk cells.

Topics: Animals; Cattle; Cytokines; Dairying; DNA Primers; Female; Gene Expression; Interleukin-1; Interleukin-6; Lactoferrin; Leukocytes; Lipopolysaccharide Receptors; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Milk; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

Pathogenic microorganisms invading the mammary gland induce an inflammatory reaction which includes an increase of somatic cells in milk and activation of bacteriostatic enzymes and proteins in milk. During spontaneously occurring subclinical mastitis the somatic milk cells, mainly macrophages, secrete cytokines, eicosanoids, acute phase proteins and other immunomediators. In contrast, the bacteriostatic protein lactoferrin is mainly secreted by mammary epithelial tissue, while major milk proteins like alpha-lactalbumin and kappa-casein are down-regulated already during subclinical infection. Changes of the mRNA expression of various immunomediators in the mammary tissue of cows during 12 h after induction of mastitis via intramammary administration of lipopolysaccharide (LPS) in several studies are reported. Six healthy lactating cows were injected in one quarter with 100 microg Escherichia coli-LPS (O26: B6) and the contralateral quarter with saline (9 g/l) serving as control. mRNA expression in mammary biopsy samples of various inflammatory factors and milk proteins at 0, 3, 6, 9 and 12 h after LPS administration was quantified by real-time reverse transcription-PCR. In LPS-challenged quarters tumour necrosis factor alpha and cyclooxygenase-2 mRNA expression increased to their highest values (P<0.05) at 3 h after LPS-challenge. Expression of lactoferrin, lysozyme, inducible nitric oxide synthase, and of the apoptotic factors caspase-3, caspase-7 and FAS was elevated (P<0.05) and peaked at 6 h after challenge. No significant increase in mRNA expression of platelet-activating factor acethylhydrolase, 5-lipoxygenase, and insulin-like growth factor 1 was found. None of the parameters tested did change significantly in the control quarters. mRNA expression of major milk proteins did not change significantly in response to the LPS challenge (alphaS1-casein, alphaS2-CN, beta-CN and beta-lactoglobulin) except for alpha-lactalbumin which decreased (P<0.05) in LPS-treated and control quarters and for kappa-CN which decreased in the LPS-treated quarters. In conclusion, mRNA expression of the majority albeit not all inflammatory factors changed within hours of LPS challenge. Decreased gene expression of alpha-lactalbumin and kappa-CN may reduce milk yield and suitability for cheese production.

Topics: Animals; Caspase 3; Caspase 7; Caspases; Cattle; Cyclooxygenase 2; Escherichia coli; Fas Ligand Protein; Female; Gene Expression; Immunity; Lactoferrin; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Membrane Glycoproteins; Muramidase; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tumor Necrosis Factor-alpha

Mastitis is a major cause of economic loss to the dairy industry. Lactoferrin (Lf) is known to contribute to resistance against bacterial infections. Hence, we decided to characterize the relevance between mastitis resistance and the variants of Lf gene. By using PCR-SSCP, five fragments within 5' region and all exons of bovine lactoferrin gene were amplified and identified the nucleotide diversity. For the five segments within the 5'-region: Lf5'-1, Lf5'-2, Lf5'-3, Lf5'-4, and Lf5'-5 from upstream to downstream, we found that three had base variation. Totally, mutations were observed in Lf5'-1, Lf5'-3, and Lf5'-5, exons 4, 8, 9, 11, 15, and intron 4. We analyzed the effects of all mutated loci on milk production traits with least squares method.

Topics: Animals; Base Sequence; Cattle; Cell Count; DNA; Female; Lactoferrin; Least-Squares Analysis; Mastitis, Bovine; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Sequence Alignment

Mammary gland epithelial cells are likely to be important effectors in defending against mastitis, yet little is known about their response mechanisms. Here, we describe a cryopreserved bovine mammary epithelial cell model to study the infection response. Primary cell cultures from four Holstein cows were prepared, and frozen after two passages. The cell cultures from each cow were then thawed and maintained separately, yet simultaneously, and exposed to treatments that included infection with Staphylococcus aureus or exposure to LPS from Escherichia coli. A clear inflammatory response was shown by a significant (P < 0.05), dose dependent, increase of lactoferrin and IL-8 secretion within 24h in response to S. aureus or LPS. Marked increases (P < 0.05) in lactoferrin, TNF-alpha and serum amyloid A (SAA) mRNA expression were also observed. The results indicate the usefulness of our model to study infection responses of mammary epithelial cells, where all cells are simultaneously exposed to the same infection pressure. These responses can be studied over time, and most importantly, biological replication is provided by the four different genotypes being investigated individually. Finally, the results indicate that mammary epithelial cells play an important role in inflammatory response, through the production of pro-inflammatory cytokines, an acute phase protein, and lactoferrin.

Topics: Animals; Apolipoproteins; Blotting, Northern; Cattle; Cell Culture Techniques; Cryopreservation; Epithelial Cells; Female; Interleukin-8; Lactoferrin; Lipopolysaccharides; Mastitis, Bovine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Amyloid A Protein; Staphylococcal Infections; Staphylococcus aureus

Antibacterial effects of bovine lactoferrin were studied in vitro against microorganisms isolated from mastitic milk in Tokachi area, Hokkaido, Japan. Microorganisms isolated were Escherichia coli (11 isolates), Klebsiella pneumoniae (5 isolates), enterococci (8 isolates), Staphylococcus aureus (10 isolates), coagulase negative staphylococci (CNS, 13 isolates), streptococci (11 isolates), Prototheca zopfii (7 isolates) and yeast-like fungi (9 isolates). Lactoferrin has been known as a multifunctional protein and its antimicrobial effect is one of the most essential function of it. In order to compare their susceptibilities against lactoferrin, the minimal inhibitory concentration values were estimated by a microplate assay method using 96-well microplate, which involved measuring the optical density of the cultures. Prototheca zopfii was highly sensitive to bovine lactoferrin and complete inhibition of this microorganism was observed even at the low concentration of 7 mug/ml. On the other hand, E. coli and enterococci showed resistance against lactoferrin action and staphylococci showed strain-dependent resistance.

Topics: Animals; Bacteria; Cattle; Female; Fungi; Japan; Lactoferrin; Mastitis, Bovine; Microbial Sensitivity Tests; Milk

The concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens. Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarter milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test. Lf concentrations (means +/- standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 +/- 0.39 and 2.70 +/- 0.39, respectively. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows. The mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05). The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01). The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods. Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score. Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis.

Topics: Aging; Animals; Cattle; Cell Count; Corynebacterium; Corynebacterium Infections; Female; Lactation; Lactoferrin; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus aureus

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.

Topics: Animals; Anti-Infective Agents; Cattle; Colony Count, Microbial; Drug Therapy, Combination; Female; Lactoferrin; Mastitis, Bovine; Mice; Neutrophils; Penicillin G; Phagocytosis; Random Allocation; Staphylococcal Infections; Staphylococcus aureus

To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS).. 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis.. Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL.. In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner.. Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cattle; Female; Glycyrrhizic Acid; Histamine; Infusions, Parenteral; Lactalbumin; Lactoferrin; Leukocytes; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Time Factors

The therapeutic effect of administering lactoferrin hydrolysate (LFH) into the mammary glands of cows with subclinical mastitis was evaluated. Seven millilitres of a preparation of LFH (7% protein) was infused into 35 quarters of 25 cows with subclinical mastitis. The numbers of bacteria in the milk from infected quarters decreased, and bacteria disappeared by the 14th day after the administration of LFH. The mean somatic cell counts (SCC) peaked one day after administration of LFH and the counts were significantly p < 0.01) decreased on days 7, 14 and 21 compared to those before the administration of LFH. The mean lactoferrin concentration in the milk peaked on days 2 or 3 and then gradually decreased to day 14, returning to the level before the administration of LFH. It appears that administration of LFH may have a therapeutic effect when infused into the quarters of cows with subclinical mastitis.

Topics: Animals; Cattle; Cell Count; Escherichia coli Infections; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Streptococcal Infections

The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), originally isolated from bovine mastitis. Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml. Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk. We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves. Growth inhibition was seen in the broth but hardly at all in whey. The isolates of E. coli and CNS did not grow sufficiently well in whey to draw any conclusions. The most effective inhibitory activity of Lf was seen against E. coli and P. aeruginosa. All 5 E. coil isolates had similar growth patterns. Inhibition of growth by Lf was concentration-dependent. The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.

Topics: Animals; Bacteria; Cattle; Female; Lactoferrin; Mastitis, Bovine; Microbial Sensitivity Tests

Lactoferrin, lysozyme and the lactoperoxidase-thiocyanate-peroxide-system are naturally occurring antimicrobial components of milk. The objective of this study was to examine, whether these components were responsible for negative results, when mastitis milk is cultured microbiologically. Quarter milk samples from 75 cows with clinical mastitis on a dairy farm in Brandenburg were submitted for microbiological culture and analysed for the content and the activities of the three components. Animals from all stages of lactation with clinical mastitis were included in the study. Animals were examined clinically and milk samples were collected prior to first treatment. Secretions from quarters with clinical mastitis were compared to those of neighbouring quarters without clinical mastitis. Secretions with positive cultural results were compared to those with negative results. The concentrations or activities of the three factors were significantly higher in the diseased quarters than in the quarters without clinical signs of mastitis. The concentration of lysozyme increased with severity of the clinical signs (local swelling and changes in secretion). The concentration of lactoferrin was significantly higher in quarters with slight alterations of glandular tissue than in quarters with medium or severe alterations (P < 0.05). LPS-activities did not correlate with the severity of clinical signs. No differences in the concentration of lactoferrin or LPS-activities were seen between mastitis with positive and negative culture results. The concentration of lysozyme was even higher in culturally positive samples than in negative samples (P < 0.05). Results from this study indicate that the three factors examined did not impair the results of microbiological culture of milk samples from quarters with clinical mastitis.

Topics: Animals; Cattle; Female; Fermentation; Hydrogen Peroxide; Lactoferrin; Lactoperoxidase; Mastitis, Bovine; Milk; Muramidase; Thiocyanates

Staphylococcus aureus can survive in conditions of extremely low iron concentration. The ability of S. aureus to use two exogenous hydroxamate types of siderophores (desferrioxamine and ferrichrome) and four iron-containing proteins found in cattle (hemin, hemoglobin, ferritin, and lactoferrin) were tested on 16 reference and clinical isolates. For all strains tested, ferrichrome and desferrioxamine showed strong growth-promoting activities in a disk diffusion assay and in liquid medium. The heme proteins hemin and hemoglobin were also found to support growth in culture media lacking other iron sources, while lactoferrin failed to do so. On media containing the iron chelator dipyridyl, ferritin induced a growth inhibition effect that was further enhanced in the presence of lactoferrin in seven of the 13 tested strains. Staphylococcus aureus was able to bind hemin and the level of binding activity was not increased after growth in iron-rich or -poor media. Dot-blot competition tests showed that biotin-labeled lactoferrin binds to S. aureus, and this binding can be inhibited by unlabeled lactoferrin. Expression of lactoferrin-binding activity was independent of the level of iron in the medium and the iron saturation status of lactoferrin. For each strain tested, ligand blots showed lactoferrin-binding proteins of molecular weights ranging from 32 to 92 kDa. Possible functions of these lactoferrin-binding proteins could not be related to iron acquisition mechanism in S. aureus.

Topics: Animals; Cattle; Culture Media; Deferoxamine; Ethylenediamines; Female; Ferrichrome; Ferritins; Hemin; Hemoglobins; Iron; Iron Chelating Agents; Lactoferrin; Mastitis, Bovine; Staphylococcus aureus

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (UT516, UT519, ATCC 27957) were used to determine if bovine lactoferrin (Lf) binds to bacterial cells by biotin avidin binding assay (BABA), enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay. Binding assays revealed that all strains of S. dysgalactiae subsp. dysgalactiae (S. dysgalactiae) evaluated in this study bound to Lf. However, differences in Lf binding capability among strains and between methods used were detected. Binding of Lf was not inhibited by transferrin (Tf) and Lf moiety molecules (mannose, galactose, and lactose) but by Lf. This study demonstrates that S. dysgalactiae bound to bovine Lf in a specific manner.

Topics: Animals; Avidin; Binding, Competitive; Biological Assay; Biotin; Biotinylation; Cattle; Enzyme-Linked Immunosorbent Assay; Lactoferrin; Mastitis, Bovine; Streptococcal Infections; Streptococcus

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half-life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty-eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.

Topics: Animals; Cattle; Female; Injections; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk

The objective of the present study was to evaluate the therapeutic potential of bovine lactoferrin or lactoferricin in combination with penicillin G against Staphylococcus aureus. Minimal inhibitory concentrations of lactoferrin, lactoferricin, penicillin, and combinations of lactoferrin or lactoferricin with penicillin were determined for 15 S. aureus strains including several strains resistant to beta-lactam antibiotics. The fractional inhibitory concentration index indicated a synergistic effect between lactoferrin and penicillin. Combination of lactoferrin with penicillin increased the inhibitory activity of penicillin by two- to fourfold and reduced the growth rate in S. aureus strains tested, whereas the increase in the inhibitory activity of lactoferrin by penicillin was 16- to 64-fold. The addition of iron to the medium containing a combination of penicillin and lactoferrin had no effect on growth inhibition. Electron microscopy revealed that concentration below the minimal inhibitory concentrations of penicillin induced important ultrastructure alterations, which were further enhanced by the presence of lactoferrin. When S. aureus cells were grown in the presence of a combination of penicillin and lactoferrin, changes in the protein profile of the bacteria, including the disappearance of several protein bands due to the presence of lactoferrin, were observed. These data suggest that bovine lactoferrin or lactoferricin in combination with beta-lactam antibiotics can increase the antibacterial activity of these antibiotics against S. aureus resistant to antibiotics.

Topics: Animals; Bacterial Proteins; Cattle; Drug Resistance, Microbial; Drug Synergism; Female; Lactoferrin; Mastitis, Bovine; Microscopy, Electron; Penicillin G; Penicillins; Staphylococcus aureus

To determine whether lactoferrin (LF) or milk influenced adherence of Streptococcus uberis to bovine mammary epithelial cells.. Three strains of S uberis from cows with mastitis, pooled milk samples from 3 clinically healthy Jersey cows early in the lactation period, and bovine mammary epithelial cells from a clonal cell line.. Adherence of S uberis to bovine mammary epithelial cells in the presence of various concentrations of LF or milk and after pretreatment of bacteria with LF or milk was tested. Bacteria were cultured with mammary epithelial cell monolayers for 1 hour. The culture supernatant was removed, and the epithelial cells were lysed. Adherence index was calculated as number of colony-forming units (CFU) in the cell lysate divided by number of CFU in the supernatant times 10,000.. All 3 strains of S uberis were found to bind to purified LF and LF in milk. Addition of LF to the culture medium enhanced adherence of all 3 strains to mammary epithelial cells, whereas addition of milk enhanced adherence of 2 strains and decreased adherence of the third. Pretreatment of bacteria with LF or milk increased adherence of 1 of the strains but decreased adherence of the other 2. Increased adherence was antagonized by rabbit antibovine LF antibody.. Results suggest that LF may function as a bridging molecule between S uberis and bovine mammary epithelial cells, facilitating adherence of the bacteria to the cells.

Topics: Alkanes; Animals; Bacterial Adhesion; Blotting, Western; Cattle; Colony Count, Microbial; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Female; Fluorescein-5-isothiocyanate; Immune Sera; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Rabbits; Streptococcal Infections; Streptococcus; Surface Properties

The activity of novobiocin against Escherichia coli ATCC 25922 and three E. coli strains that were isolated from cases of bovine mastitis was determined in timekill studies in the presence of bovine lactoferrin. Lactoferrin alone did not affect the growth of any of the strains of E. coli. A combination of 1.0 mg/ml of lactoferrin and novobiocin at 1/16x minimum inhibitory concentration (MIC) was bactericidal for E. coli ATCC 25922. When the concentration was increased to 3.0 mg/ml of lactoferrin, novobiocin was bactericidal at 1/64x MIC. Among the mastitis strains tested, 6789 and 6806 were more susceptible to killing by novobiocin than was strain 6800. Strains 6789 and 6806 were killed when treated with novobiocin concentrations of 2, 1/2, and 1/4x MIC. When these strains were also treated with lactoferrin at 3.0 mg/ml, there was a bacteriostatic effect at novobiocin concentrations of 1/8 and 1/16x MIC for strains 6789 and 6800. Strain 6806 appeared to be more susceptible to the combination of lactoferrin and novobiocin as was evidenced by a bactericidal effect over the 24-h testing period. The combination treatment with cephapirin and lactoferrin showed that there was a synergistic bactericidal effect against all of the E. coli strains tested. These studies indicate that lactoferrin can potentiate the activity of antibiotics against Gram-negative bacteria.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cephalosporins; Cephapirin; Drug Synergism; Escherichia coli; Female; Lactoferrin; Mastitis, Bovine; Microbial Sensitivity Tests; Novobiocin

All strains of Streptococcus uberis evaluated bound to lactoferrin (Lf) in milk as detected by polyacrylamide gel electrophoresis and Western blotting. A biotin-avidin-based microplate binding assay and ELISA also revealed that these bacterial strains bound to purified Lf. Binding of bacteria of Lf was not inhibited by mannose and galactose, indicating that glycosidic domains of the Lf molecule were not involved in binding. Lf binding was also unaffected by bovine transferrin. Western blot analysis demonstrated that there were at least two bacterial proteins involved in Lf-binding. Lf binding by S. uberis could enable this bacterium to acquire iron necessary for its growth.

Topics: Animals; Biological Assay; Blotting, Western; Cattle; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Lactoferrin; Mastitis, Bovine; Milk; Streptococcus

The lactoferrin (LF) concentration in the milk from dairy cows with clinical mastitis was determined to evaluate the relationship between the LF concentration (LFC) in milk and the non-specific defensive capability of the udder. The mean LFC in 368 milk samples from 319 cows with clinical mastitis was significantly higher (p < 0.01) than that of normal cows. The mean LFC in milk from quarters infected with Mycoplasma bovis or Staphylococcus aureus was significantly higher (p < 0.05) than that of quarters infected with coagulase-negative staphylococci (CNS). In Escherichia coli mastitis, the level of LFC in milk from cows with peracute mastitis was significantly lower (p < 0.01) than that from cows with acute mastitis. In cases of mastitis due to E. coli, the mean LFC in milk from cows that needed more than 10 days to recover from the mastitis or were not cured was significantly lower (p < 0.05) than that for cows which took less than 10 days to be cured. The mean LFC in milk from cows with peracute E. coli mastitis was significantly lower (p < 0.05) than that for cows with mastitis associated with environmental streptococci or CNS, although these low LF levels were somewhat increased after 46 h from the occurrence of mastitis. These results suggest that the decreased levels of LF in peracute E. coli mastitis may be associated with the progress of inflammation in the early phase of mastitis.

Topics: Animals; Cattle; Escherichia coli; Escherichia coli Infections; Female; Immunodiffusion; Lactoferrin; Mastitis, Bovine; Milk; Mycoplasma; Mycoplasma Infections; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus

Bovine lactoferrin is an iron-binding protein present in mammary gland secretions. The exposure of Streptococcus agalactiae to bovine lactoferrin resulted in the binding of this protein to all the 12 strains of bovine origin tested, and also, although to a lesser degree, to the five tested strains of human origin. The interaction of lactoferrin with one high-binding bovine strain (24/60, the prototype NT/X strain) was studied. Binding was time-dependent, dose-dependent, and saturable. The binding of lactoferrin was slightly affected by cultivation conditions, and appeared to be heat-stable. The binding of biotinylated lactoferrin was inhibited by unlabelled lactoferrin but not by bovine serum albumin.

Topics: Animals; Binding, Competitive; Cattle; Female; In Vitro Techniques; Kinetics; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Protein Binding; Streptococcal Infections; Streptococcus agalactiae

Immunoglobulins (Ig) and antibacterial proteins like lysozyme and lactoferrin are components of the humoral defence against infections. Changes in Ig, lysozyme and lactoferrin concentrations during endotoxin-induced inflammation in the test cistern and udder quarter of the dry cow were studied. Surgical closure of the passage between teat and udder cisterns enabled studies of reactions in the teat cistern without interference of the mammary gland. After endotoxin infusion, IgG1, IgG2, lysozyme, and to some extent IgM, increased in the teats and udder quarters, and were positively correlated with changes in somatic cell counts. No significant changes were observed in IgA or lactoferrin. The origin and significance of Ig, lysozyme and lactoferrin in the bovine teat and udder are discussed. Ig probably originated both from serum and from local plasma cells, while leukocytes appeared to be the source of lysozyme during inflammation. Secretory epithelium appeared to be the source of lactoferrin. Support for this theory was the almost total absence of lactoferrin in teat cistern samples.

Topics: Animals; Cattle; Endotoxins; Female; Immunoglobulins; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Muramidase

Topics: Animal Experimentation; Animals; Animals, Genetically Modified; Cattle; DNA; Genes; Genetic Engineering; Humans; Industry; Lactoferrin; Mastitis, Bovine; Netherlands

A total of 103 Staphylococcus aureus strains isolated from bovine mastitis were tested for bovine lactoferrin binding in a 125I-labeled protein binding assay. More than 85% of the strains demonstrated high to moderate and a few showed little or no binding. Bovine lactoferrin binding to S. aureus cells was high when grown on blood, nutrient, or proteose-peptone agar, but the binding capacity was low with cells grown on salt rich media, in skim milk, or in broth. The kinetics of 125I-labeled bovine lactoferrin binding required approximately 90 min for complete saturation with optimal interaction in the pH range 4.0 to 7.0. The lactoferrin-staphylococci interaction was specific with a high affinity (association constant, Ka 14 x 10(6) L/mol). Scatchard plot analysis estimated the number of binding sites per cell at 7200 on strain SA-340. Unlabeled bovine lactoferrin effectively displaced the binding of the labeled ligand to strain SA-340 in a dose-dependent manner. Bovine lactoferrin binding was inhibited or displaced by human lactoferrin. Various plasma, connective tissue, or mucosal secretory proteins tested did not inhibit lactoferrin-staphylococci interaction. Bovine lactoferrin binding components on SA-340 were resistant to glycolytic enzymes and moderately susceptible to proteolytic digestion. Two proteins with an estimated molecular weight of approximately 92 and 67 kDa were identified as bovine lactoferrin binding components of S. aureus strain SA-340.

Topics: Animals; Binding, Competitive; Cattle; Culture Media; Lactoferrin; Mastitis, Bovine; Receptors, Cell Surface; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus aureus strains (n = 100) isolated from bovine mastitis were classified according to the presence of capsular polysaccharide serotype 5 (n = 46), type 8 (n = 26), and non-5/8 (n = 28). Strains from each type were tested for protein interaction in a 125I-labeled ligand binding assay. A majority of type 5 and type 8 strains showed a higher degree of binding to lactoferrin, fibronectin, and IgG than the non-5/8 strains. Fibrinogen binding was low in all serotypes. Most of the type 5 and non-5/8 strains bound less than 10% laminin, whereas type 8 strains bound laminin in the 11 to 20% range. Non-5/8 strains significantly differed from type 5 in lactoferrin, fibronectin, fibrinogen, and IgG and also from type 8 in fibrinogen and IgG binding. The differences in protein binding between type 5 and type 8 were nonsignificant. The degree of lactoferrin binding in all types positively correlated with laminin binding. Lactoferrin and fibrinogen bindings were correlated in type 5 and type 8 strains. Lactoferrin and fibronectin bindings were correlated only in type 5 strains. These data suggest that bovine lactoferrin binding is common and associated with subepithelial matrix protein interactions in certain serotypes of S. aureus.

Topics: Animals; Cattle; Extracellular Matrix Proteins; Female; Fibrinogen; Fibronectins; Immunoglobulin G; Lactoferrin; Laminin; Mastitis, Bovine; Protein Binding; Serotyping; Staphylococcal Infections; Staphylococcus aureus

Healthy, midlactation cows were given intramammary infusions of 10 micrograms of endotoxin in two homolateral quarters. Productive, inflammatory, and systemic responses were studied to investigate the pathophysiological effects of mastitis on lactational performance. Endotoxin suppressed milk yield in all quarters of treated cows. A more severe and prolonged suppression occurred in infused quarters compared with uninfused quarters. The fat percentage of milk from all quarters was increased with a greater increase occurring in infused quarters. The protein composition of milk was elevated, and the lactose concentration was depressed in infused quarters. Mammary inflammation--as measured by milk SCC, NAGase, serum albumin, and lactoferrin--was limited to infused quarters. Changes in milk NAGase closely paralleled changes in milk SCC. Daily feed intake was unaffected, and serum glucose levels did not decline following infusion. The lactose concentration of urine increased rapidly after infusion. Reduction in milk yield in all quarters, but varying changes in milk composition in infused versus uninfused quarters suggest that mastitic hypogalactia is mediated by multiple pathophysiological events and is not solely due to inflammatory damage to the mammary epithelium. Part of the reduced lactational performance may result from escape of milk components from the udder into the circulation.

Topics: Acetylglucosaminidase; Animals; Blood Glucose; Cattle; Cell Count; Disease Models, Animal; Eating; Endotoxins; Female; Lactation Disorders; Lactoferrin; Lactose; Lipids; Mastitis, Bovine; Milk; Milk Proteins; Serum Albumin, Bovine

Midlactation cows were infused with 10 micrograms endotoxin in the same two homolateral quarters after each of several consecutive milkings to study the effect of prolonged, endotoxin-induced mastitis on lactational performance. The initial infusion induced an acute response with systemic involvement. Inflammation developed in infused quarters, and milk production declined and milk composition was altered in all quarters. Subsequent infusions failed to induce systemic responses. Furthermore, milk yield and composition in uninfused quarters returned to pre-treatment levels despite further infusions. In infused quarters, milk yield, protein percentage and serum albumin concentration showed partial recovery during the endotoxin infusion period. In contrast, decreased lactose concentration, and increased cell count, N-acetyl-beta-D-glucosaminidase and lactoferrin levels persisted throughout the infusion period. After infusions were stopped, all measurements returned to near pretreatment levels. These data demonstrate that systemic, but not local, responses become refractory to multiple intramammary endotoxin infusions, and that multiple infusions have continued but little progressive or permanent, inhibitory effects on lactational performance despite a persistent mammary leucocytosis.

Topics: Acetylglucosaminidase; Animals; Cattle; Cell Count; Endotoxins; Escherichia coli; Female; Lactation; Lactation Disorders; Lactoferrin; Lactose; Lipids; Mastitis, Bovine; Milk; Milk Proteins; Serum Albumin, Bovine

Gram-negative bacteria (n = 192) isolated from infected bovine mammary glands were tested for growth in a pooled source of dry cow secretion. Growth of Klebsiella pneumoniae in dry cow secretion was greater than growth of Escherichia coli and Klebsiella oxytoca. Escherichia coli originating during the early dry period exhibited greater growth in dry cow secretion than those originating around calving or during lactation. Klebsiella pneumoniae growth did not differ with time of origin of intramammary infection. Escherichia coli, K. oxytoca, and K. pneumoniae growth in a synthetic medium was reduced by apolactoferrin plus Ig. Growth reduction was greatest for E. coli. Citrate reversed growth inhibition. The inhibitory properties of dry cow secretion for E. coli may contribute to the low number of naturally occurring intramammary infections originating during the early part of the dry period. Inhibitory properties of dry cow secretion are partially explained by lactoferrin acting in conjunction with antibody to prevent iron acquisition by many gram-negative bacteria.

Topics: Animals; Apoproteins; Cattle; Citrates; Culture Media; Enterobacter; Escherichia coli; Female; Gram-Negative Bacteria; Immunoglobulins; Iron; Klebsiella; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Serratia

Bovine lactoferrin (BLf), an acute-phase iron-binding secretory protein present in secretions of the bovine udder, was demonstrated to bind to the following staphylococcal species associated with bovine intramammary infections: S. epidermidis, S. warneri, S. hominis, S. xylosus, S. hyicus, and S. chromogenes. The degree of 125I-labeled BLf uptake significantly varied among the blood agar-grown cells of all six species of coagulase-negative staphylococci tested. Isolates identified as S. xylosus demonstrated the highest (mean, 35.1 x 10(6) +/- 13.3 x 10(6) nmol) and S. hyicus the lowest (mean, 10.7 x 10(6) +/- 5.9 x 10(6) nmol) binding to 125I-BLf. BLf binding was optimum at an acidic pH, with time-dependent binding saturation ranging from 70 min for S. warneri to 240 min for S. hominis. The BLf-binding mechanism was specific, with affinity constants (Ka values) ranging between 0.96 x 10(6) and 11.90 x 10(6) liters/mol. The numbers of BLf-binding sites per cell, as determined by using Scatchard analysis, were as follows: S. epidermidis, 3,600; S. warneri, 1,900; S. hominis, 4,100; S. xylosus, 4,400; S. hyicus, 6,100; and S. chromogenes, 4,700. 125I-BLf binding to all species was inhibited by unlabled BLf and unlabeled human lactoferrin, whereas none of the various plasma, connective tissue, or mucosal secretory proteins or carbohydrates tested caused significant interference. BLf-binding receptors of the six coagulase-negative staphylococcal species demonstrated marked differences in patterns of susceptibility to proteolytic or glycolytic enzyme digestion and to heat or periodate treatment. These data suggest that the BLf-binding components in S. epidermidis and S. warneri are proteins containing glycosidyl residues. In the remaining four species, the proteinaceous nature of the BLf-binding component was evident, but the involvement of glycosidyl residues was not clear. Results of this study establish the presence of specific binding components for BLf on coagulase-negative staphylococci isolated from bovine intramammary infections.

Topics: Animals; Bacterial Proteins; Binding Sites; Carrier Proteins; Cattle; Cell Membrane; Coagulase; Female; Kinetics; Lactoferrin; Mastitis, Bovine; Species Specificity; Staphylococcal Infections; Staphylococcus

Microbiological methods for detection of antibiotic residues in milk give no explanations regarding the identity of the inhibitory substance(s). Natural antibacterial substances, present at higher concentrations in mastitic milk and in colostrum, occasionally cause false positive results in antibiotic assays. In an earlier investigation, lysozyme and lactoferrin were shown to inhibit the growth of Bacillus stearothermophilus var. calidolactis spores, used as test organism in Delvotest P. To study the effect of high lysozyme and lactoferrin concentrations in milk on the Delvotest P, cows were subjected to acute experimental mastitis by infusion of Salmonella typhimurium SH 4809 endotoxin. Milk samples were collected up to 11 h postinfusion. Concentrations of lactoferrin and lysozyme, somatic cell count, and effect on Delvotest P were determined. A positive reaction in the Delvotest correlated well with an increase in lactoferrin and lysozyme concentrations. The nature of the inhibitory effect is briefly discussed.

Topics: Acute Disease; Animals; Cattle; Cell Count; Female; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Muramidase

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.

Topics: Animals; Cattle; Cattle Diseases; Escherichia coli; Escherichia coli Infections; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk

Cell-free, fat-free mammary secretions were tested in vitro for ability to support growth of streptococci associated with mastitis. Secretions were obtained prior to drying off, during the dry period, at calving, and during lactation from four cow treatment groups. Treatment groups were dry cow therapy, dry cow therapy and mammary glands subjected to induced inflammation 7 d post-drying-off, no dry cow therapy and no induced inflammation, no dry cow therapy but mammary glands subjected to induced inflammation. Growth of Streptococcus uberis, Streptococcus faecalis, and Streptococcus agalactiae in secretions from nonlactating glands was unaffected by induced inflammation. Growth of Streptococcus bovis was significantly inhibited in secretion obtained 14 d after induced inflammation. Dry cow therapy had no effect on streptococcal growth in secretion obtained 7 d after therapy. Streptococcal growth was greatest in secretions from involuted glands, and there was little or no evidence for growth inhibitory factors in cell-free, fat-free secretions obtained during the dry period. Milk from lactating glands inhibited streptococcal growth, and the inhibitory factor was presumptively identified as lactoperoxidase. Apolactoferrin, immunoglobulin, or both had little effect on streptococcal growth.

Topics: Animals; Apoproteins; Bacteriological Techniques; Body Fluids; Cattle; Citrates; Citric Acid; Culture Media; Disease Susceptibility; Female; Immunoglobulins; Inflammation; Lactation; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Novobiocin; Pregnancy; Streptococcal Infections; Streptococcus

Topics: Animals; Cattle; Female; Immunoglobulin G; Immunoglobulin M; Lactation; Lactoferrin; Lymphocyte Activation; Mastitis, Bovine; Milk; Neutrophils; Phagocytosis; Pregnancy; Serum Albumin, Bovine

An in vitro microassay was developed to evaluate antimicrobial properties of bovine apo-lactoferrin. The growth of coliform, staphylococcal, and streptococcal bacterial strains in a defined synthetic medium was inhibited by bovine apo-lactoferrin (.5 to 30.0 mg/ml). Addition of iron-saturated lactoferrin to the synthetic medium did not inhibit growth of test strains. Inhibition by apo-lactoferrin was greater for coliform than Gram-positive strains for all concentrations of apo-lactoferrin evaluated. No concentration of apo-lactoferrin proved bactericidal for either coliform or Gram-positive strains. Inhibition of two coliform strains by apo-lactoferrin (10 mg/ml) was abolished by addition of ferric iron to the assay system, indicating an iron-dependent nature of apo-lactoferrin induced inhibition of bacteria. Bicarbonate supplementation of the growth system containing apo-lactoferrin (1 mg/ml) increased inhibition of three coliform strains by apo-lactoferrin. Addition of increasing concentrations of citrate (2.0 mg/ml) to an assay system containing apo-lactoferrin (5 mg/ml) resulted in a concomitant reduction of growth inhibition of three coliform strains. These data indicate a potential relationship between the molar ratio of citrate to lactoferrin of the lacteal secretion and its capacity to inhibit coliform strains associated with mastitis.

Topics: Animals; Apoproteins; Bicarbonates; Cattle; Citrates; Citric Acid; Culture Media; Escherichia coli; Female; Iron; Klebsiella pneumoniae; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Sodium Bicarbonate; Staphylococcus aureus; Streptococcus

Activity of N-acetyl-beta-D-glucosaminidase was measured in foremilk samples collected from cows. A total of 64 samples were collected from these cows, some of which were affected by mastitis. They were examined for N-acetyl-beta-D-glucosaminidase activity, cell count in whole milk, and milk chloride, lactose, and lactoferrin. The correlation coefficient was .72 between activity and cell count, -.80 between activity and lactose, and .88 between activity and chloride. Therefore, the correlations between N-acetyl-beta-D-glucosaminidase activity and milk components, chloride and lactose, were larger than that between N-acetyl-beta-D-glucosaminidase activity and cell count. The correlation coefficient was high (.91) between N-acetyl-beta-D-glucosaminidase and lactoferrin. The N-acetyl-beta-D-glucosaminidase activity in cow's milk has potential for diagnosis of mastitis.

Topics: Acetylglucosaminidase; Animals; Cattle; Cell Count; Chlorides; Clinical Enzyme Tests; Female; Hexosaminidases; Lactoferrin; Lactoglobulins; Lactose; Mastitis, Bovine; Milk

Two immunised and three unimmunised cows were infected in a single mammary gland with 10(4) CFU of the vaccine Escherichia coli strain. Immunisation comprised systemic (subcutaneous) injection of killed bacteria at drying-off and one intramammary infusion five weeks later. Immunoglobulin (Ig) G1, IgG2, lgM, serum albumin (BSA) and lactoferrin concentrations were monitored by sampling the inoculated glands at 2 h-intervals during the first 16 h post-inoculation, then at each milking for four days. Whether immunised or not, mammary glands started to react at 10 h post-inoculation. During the early acute phase, IgG1 and IgG2 permeated from blood into milk at a rate similar to BSA. Later on, IgM (and at a lower degree IgG1) concentrations were higher than expected on the basis of passive transfer. Marked protein exudation was seen in all of the cows but one. Nevertheless, this cow (immunised) showed an intense cellular reaction like the other animals. Lactoferrin concentrations rose from 24-32 h post-inoculation and remained elevated to the end of the observed period in inoculated quarters in unimmunised cows. By contrast, in immunised cows lactoferrin concentrations remained low. Heat-labile bactericidal activity against a serum-sensitive E. coli strain appeared concomitantly with rise in BSA concentration. Heat-resistant bactericidal activity of cell-free milk was detected one or two days later in three of the cows. Bacteriological cure of quarters occurred without therapy in all cases.

Topics: Animals; Bacterial Vaccines; Cattle; Escherichia coli Infections; Female; Immunization; Immunoglobulin G; Immunoglobulin M; Lactoferrin; Lactoglobulins; Mammary Glands, Animal; Mastitis, Bovine; Milk; Milk Proteins; Serum Albumin, Bovine; Time Factors

Topics: Animals; Antibodies, Bacterial; Cattle; Complement System Proteins; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Mastitis, Bovine; Muramidase; Properdin; Vaccination

Topics: Animals; Cattle; Citrates; Female; Lactation; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Pregnancy; Staphylococcal Infections; Staphylococcus aureus

Mean transferrin and lactoferrin concentrations in whey samples from 376 uninfected quarters of 42 Holstein X Friesian cows on the 30th, 150th and 270th days of lactation were respectively 0.030 and 0.080 mg/ml. The mean transferrin concentration in serum was 4.63 mg/ml. Lactation number and location of quarters did not influence milk lactoferrin and transferrin values. Lactoferrin concentration increased significantly (P less than 0.01) in uninfected quarters from 0.03 to 0.06 and to 0.15 mg/ml on the three successive sampling times. Transferrin whey concentration increased significantly (P less than 0.01) only in late lactation, from 0.025 (30th and 150th days of lactation) to 0.035 mg/ml (270th day of lactation). Lactoferrin concentration increased significantly (P less than 0.01) in quarters infected by major pathogens (41 samples) whereas minor pathogen infections (61 samples) caused no significant increase. The correlation coefficients between milk lactoferrin and transferrin concentrations and somatic cell-count were 0.42 and 0.65 respectively.

Topics: Age Factors; Animals; Cattle; Female; Lactation; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Pregnancy; Transferrin

Analyses were of lactoferrin and mastitis of milk samples taken every other month from 830 Holstein cows in eight herds for 1 yr. Patterns of variation and amounts in lactation, colostrum, and dry period were similar to reports. In negative mastitis tests of milk samples, lactoferrin content was lower during lactation and lower as age of the cow advanced than for positive tests. However, heritability of mastitis was .14, whereas for lactoferrin was .44 in records of 289 cows. This latter is high enough to be useful but unreliable with large standard error .30. Several sire groups differed significantly.

Topics: Animals; Cattle; Female; Lactation; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Pregnancy

Topics: Animals; Cattle; Female; Lactoferrin; Lactose; Mammary Glands, Animal; Mastitis, Bovine; Milk

An experimentally induced Escherichia coli infection of a bovine mammary gland resulted in a 30-fold increase in lactoferrin (Lf) concentration in the mammary secretion by 90 h postinoculation and a 4-fold increase in total daily production of Lf by 264 h postinoculation in the infected quarter. A simultaneous rise and fall of bovine serum albumin (BSA) and immunoglobulin G (IgG) concentrations occurred during the acute phase of the infection. Peak BSA and IgG levels were reached 36 h before peak Lf levels. BSA concentrations declined rapidly after the acute phase, whereas IgG and Lf levels remained elevated and decreased slowly as the infection subsided. A decline in alpha-lactalbumin concentration by 48 h postinoculation indicated decreased synthetic capability. The increased Lf production may be a result of a specific response of secretory tissue to inflammatory agents and thus the infectious process. Analogous changes in Lf, IgG, and BSA were observed during a natural coliform infection. Sephadex G-200 chromatography of mastitis skim milk showed that Lf approximated the monomer (molecular weight 77,100) early in infections progressed and abated, the apparent molecular weight of Lf increased to approximately that of the trimer and subsequently decreased to about 1.5 times that of the monomer.

Topics: Acute Disease; Animals; Cattle; Cell Count; Escherichia coli Infections; Female; Immunoglobulin G; Lactalbumin; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Molecular Weight; Serum Albumin, Bovine

Bovine apo-lactoferrin (apo-Lf) was added to in vitro cultures of eight strains of coliform bacteria associated with bovine mastitis. As little as 0.02 mg of APO-Lf per ml resulted in marked inhibition of growth of all coliforms. Growth inhibition was lost if saturated Lf or iron plus apo-Lf was added to the synthetic medium. The inhibition of growth increased as the concentration of apo-Lf increased from 0.02 to 0.2 mg/ml for Klebsiella pneumoniae (OARDC-A1), Klebsiella spp. (K1-21), and Aerobacter aerogenes (55-12222) and 2 mg/ml for A. aerogenes (76-2414-1), Escherichia coli (60-Lilly), E. coli (66-S16), and Klebsiella spp. (K6-24). As the concentration of apo-Lf was increased above 0.2 or 2 mg/ml, there was less inhibition of growth except for E. coli (33-C4). Apo-Lf at 20 mg/ml was bactericidal for E. coli (33-C4). Results are compatible with the hypothesis that coliform bacteria respond to low-iron environments by production of iron-sequestering agents that complete effectively with apo-Lf for free iron. Addition of apo-Lf plus citrate resulted in loss of growth inhibition. The molar ratio (citrate to apo-Lf) was found to be more important than the absolute concentration of either component. A ratio of 75 resulted in 50% growth inhibition, whereas ratios of 300 and greater resulted in less than 10% growth inhibition. These results suggest that the ratio of citrate to Lf would be important in evaluating Lf as a nonspecific protective factor of bovine mammary secretions.

Topics: Animals; Cattle; Citrates; Culture Media; Dose-Response Relationship, Drug; Enterobacter; Enterobacteriaceae; Escherichia coli; Iron; Klebsiella pneumoniae; Lactoferrin; Lactoglobulins; Mastitis, Bovine

The mean lactoferrin (Lf) concentration determined by electroimmunodiffusion (EID) assay of whey preparations from 80 quarters of 20 normal lactating cows was 0.35 mg/ml. The mean alpha-lactalbumin (alpha-LAC) and bovine serum albumin (BSA) concentrations were 2.01 mg/ml and 0.29 mg/ml, respectively. The mean was significantly related to cell count (P smaller than 0.01), BSA (P smaller than 0.05), stage of lactation (P smaller than 0.05), and milk production (P smaller than 0.05). The Lf-milk production relationship was the only negative correlation. In 11 cows with mastitis, there was a significant (P smaller than 0.01) increase in mean Lf concentration in infected quarters from 0.55 mg/ml on day 1 of the infection to 1.89 mg/ml by day 3. By day 15 clinical signs had subsided and mean Lf concentrations had decreased to near day 1 values. On day 3 quarters infected with coliform bacteria (clinical mastitis generally more severe) had mean Lf values more than twofold greater than those quarters infected with species of Staphylococcus or Streptococcus (milder clinical signs). Noninfected (control) quarters of cows having coliform bacteria-infected quarters had slightly increased mean Lf concentrations, where Lf concentration in contral quarters of cows having quarters infected with gram-positive organisms remained unchanged.

Topics: Animals; Cattle; Electrophoresis, Polyacrylamide Gel; Escherichia coli Infections; Female; Lactalbumin; Lactation; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Pregnancy; Serum Albumin, Bovine; Staphylococcal Infections; Streptococcal Infections

Topics: Animals; Cattle; Escherichia coli; Female; Immunity; Immunoglobulins; Lactation; Lactoferrin; Leukocyte Count; Mammary Glands, Animal; Mastitis, Bovine; Milk; Muramidase; Peroxidases; Pregnancy; Thiocyanates