lactoferrin and Escherichia-coli-Infections

Escherichia coli O157:H7 shed by clinically healthy ruminants has been linked to hemorrhagic colitis and the hemolytic uremic syndrome in humans. The bacteria are spread mainly by contaminated food and water, contact with animals carrying the organisms, and person-to-person contact. Although many intervention strategies have been studied to reduce E. coli O157:H7 carriage in ruminants and its spread into the environment, none of the available methods can completely eliminate the infection. Therefore, there is need for new intervention strategies which will effectively reduce E. coli O157:H7 prevalence. Lactoferrin, a member of the transferrin protein family, is an iron-binding glycoprotein that is found in many exocrine secretions, including milk, tears, saliva, and serum. Lactoferrin has a number of biological functions including antimicrobial and immunomodulatory effects. This review summarizes latest data on the antimicrobial effect of lactoferrin against E. coli O157:H7 in in vitro and in vivo studies.

Topics: Animals; Escherichia coli Infections; Escherichia coli O157; Humans; Lactoferrin

Bacterial infections, with the most severe form being sepsis, can often not be treated adequately leading to high morbidity and lethality of infected patients in critical care units. In particular, the increase in resistant bacterial strains and the lack of new antibiotics are main reasons for the worsening of the current situation, As a new approach, the use of antimicrobial peptides (AMPs) seems to be promising, combining the ability of broad-spectrum bactericidal activity and low potential of induction of resistance. Peptides based on natural defense proteins or polypeptides such as lactoferrin, Limulus anti-lipopolysaccharide factor (LALF), cathelicidins, and granulysins are candidates due to their high affinity to bacteria and to their pathogenicity factors, in first line lipopolysaccharide (LPS, endotoxin) of Gram-negative origin. In this review, we discuss literature with the focus on the use of AMPs from natural sources and their variants as antibacterial as well as anti-endotoxin (anti-inflammatory) drugs. Considerable progress has been made by the design of new AMPs for acting efficiently against the LPS-induced inflammation reaction in vitro as well as in vivo (mouse) models of sepsis. Furthermore, the data indicate that efficient antibacterial compounds are not necessarily equally efficient as anti-endotoxin drugs and vice versa. The most important reason for this may be the different molecular geometry of LPS in bacteria and in free form. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antigens, Differentiation, T-Lymphocyte; Antimicrobial Cationic Peptides; Arthropod Proteins; Disease Models, Animal; Drug Design; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Infections; Humans; Lactoferrin; Lipopolysaccharides; Mice; Molecular Sequence Data; Sepsis

Neonatal sepsis remains a feared cause of morbidity and mortality in the neonatal period. Maternal, neonatal, and environmental factors are associated with risk of infection, and a combination of prevention strategies, judicious neonatal evaluation, and early initiation of therapy are required to prevent adverse outcomes. This article reviews recent trends in epidemiology and provides an update on risk factors, diagnostic methods, and management of neonatal sepsis.

Topics: Adaptive Immunity; Anti-Infective Agents; Antibodies, Monoclonal; Antifungal Agents; Bacterial Infections; Biomarkers; Blood Cell Count; C-Reactive Protein; Candidiasis; Escherichia coli Infections; Fluconazole; Genomics; Humans; Immunity, Innate; Immunoglobulins, Intravenous; Infant, Newborn; Infant, Newborn, Diseases; Lactoferrin; Polymerase Chain Reaction; Predictive Value of Tests; Proteomics; Risk Factors; Sepsis; Streptococcal Infections; Streptococcus agalactiae

Escherichia coli (E. coli) are the most common aerobic gram-negative bacilli in a normal intestinal tract. They cause most of the intra-abdominal infections, wound infections associated with abdominal surgery, and septicemia. Most of these infections are of endogenous intestinal origin. Lactoferrin (LF) is an iron-binding glycoprotein found in milk and various external secretions. This protein has been found to have a number of biological functions, including antimicrobial, anti-cancer, antioxidant, and immunomodulatory effects. Partial degradation of LF by pepsin can give rise to peptides termed lactoferricin (LFcin) with more potent antimicrobial activity. LF and LFcin have been shown to inhibit the growth of a number of pathogenic bacteria (including E. coli and antibiotic-resistant strains), fungi, and even viruses in both in vitro and in vivo studies. We previously demonstrated that both recombinant porcine LF (pLF) produced from yeast and a synthetic 20-residue porcine LFcin peptide exhibit antimicrobial activity in vitro. In one of our recent studies, we performed pathogen challenges, including pathogenic E. coli, Staphylococcus aureus and Candida albicans, of the digestive tract of a transgenic milk-fed animal model. The results showed that LF has broad spectrum antimicrobial activity in the digestive tract and protects the mucosa of the small intestine from injury. Our following study also revealed that pLF as a feedstuff additive enhances avian immunity, including antibody formation and cell-mediated immunity. All of these results suggest that LF could be a novel natural protein in the treatment and prevention of infections with E. coli or antibiotic-resistant bacteria strains.

Topics: Animals; Anti-Bacterial Agents; Escherichia coli; Escherichia coli Infections; Humans; Iron; Lactoferrin; Microbial Sensitivity Tests

Topics: Animals; Animals, Newborn; Anti-Infective Agents; Escherichia coli Infections; Humans; Infant; Infant, Newborn; Lactoferrin

The terminal rectal mucosa has been identified as the predominant colonization site of Escherichia coli O157:H7 in cattle, thus a possible intervention approach should directly target this colonization site. To determine the effect of lactoferrin on E. coli O157:H7 mucosal colonization at the rectum, five 6-month-old Holstein-Friesian calves were experimentally infected with E. coli O157:H7 and received daily rectal treatment with bovine lactoferrin. Three calves that did not receive the lactoferrin served as control group. The treatment decreased faecal shedding of E. coli O157:H7 and completely eliminated the infection in all animals (n=5) after 19 days administration. The rectal mucosa of all animals (n=5) was cleared from E. coli O157:H7 within 13 days of lactoferrin treatment. To evaluate the local immune responses, three calves treated previously with lactoferrin and three calves of the control group were re-infected when E. coli O157:H7 excretion was no longer detected. The rectal administration of lactoferrin resulted in an EspA- and EspB-specific IgA responses at the rectal mucosa. These mucosal antibodies were not detected in the animals which did not receive the lactoferrin powder. Interestingly, no serum IgA antibodies could be found in animals of the group that received the lactoferrin. These findings emphasize the ability of bovine lactoferrin to clear E. coli O157:H7 colonization in cattle, where lactoferrin may influence the local immune processes against E. coli O157:H7 infection. Thus, bovine lactoferrin treatment could be used in the field to eliminate high-level faecal excretion of E. coli O157:H7 in cattle.

Topics: Administration, Rectal; Animals; Carrier State; Cattle; Cattle Diseases; Escherichia coli Infections; Escherichia coli O157; Lactoferrin; Rectum

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

LF82, a prototype of adherent-invasive

Topics: Animals; Caco-2 Cells; Cattle; Cell Differentiation; DNA Damage; Enterocytes; Enteropathogenic Escherichia coli; Escherichia coli Infections; Humans; Lactoferrin

Lactoferricin (Lfcin) B, derived from lactoferrin in whey, has attracted considerable attention because of its multiple biological functions. Zoonotic enterohemorrhagic Escherichia coli (EHEC) O157:H7 has adverse effects on intestinal epithelial barrier function, leading to serious intestinal disease. In this study, the EHEC O157:H7-induced intestinal dysfunction model was developed to investigate the effects of Lfcin B on EHEC O157:H7-induced epithelial barrier disruption and microbiota dysbiosis. Results showed that the inflammatory infiltration indexes in the jejunum of Lfcin B-treated animals were significantly decreased. Lfcin B administration also significantly improved ZO-1 and occludin expression following O157:H7-induced injury. Finally, microbiota analysis of the cecal samples revealed that Lfcin B inhibited the O157:H7-induced abnormal increase in Bacteroides. Therefore, Lfcin B efficiently attenuated O157:H7-induced epithelial barrier damage and dysregulation of inflammation status, while maintaining microbiota homeostasis in the intestine, indicating that it may be an excellent food source for prevention and therapy of EHEC O157:H7-related intestinal dysfunction.

Topics: Administration, Oral; Animals; Bacteria; Cattle; Escherichia coli Infections; Escherichia coli O157; Gastrointestinal Microbiome; Humans; Intestinal Diseases; Lactoferrin; Male; Mice; Mice, Inbred C57BL

Urinary tract infection (UTI) is a prominent global health care burden. Although UTI is readily treated with antibiotics in healthy adults, complicated cases in immune-compromised individuals and the emerging antibiotic resistance of several uropathogens have accelerated the need for new treatment strategies. Here, we surveyed the composition of urinary exosomes in a mouse model of uropathgenic Escherichia coli (UPEC) UTI to identify specific urinary tract defense constituents for therapeutic development. We found an enrichment of the iron-binding glycoprotein lactoferrin in the urinary exosomes of infected mice. In subsequent in vitro studies, we identified human bladder epithelial cells as a source of lactoferrin during UPEC infection. We further established that exogenous treatment with human lactoferrin (hLf) reduces UPEC epithelial adherence and enhances neutrophil antimicrobial functions including bacterial killing and extracellular trap production. Notably, a single intravesicular dose of hLf drastically reduced bladder bacterial burden and neutrophil infiltration in our murine UTI model. We propose that lactoferrin is an important modulator of innate immune responses in the urinary tract and has potential application in novel therapeutic design for UTI.

Topics: Animals; Disease Models, Animal; Escherichia coli Infections; Exosomes; Extracellular Traps; Female; Humans; Immunity, Innate; Immunocompromised Host; Iron; Lactoferrin; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Urinary Bladder; Urinary Tract Infections; Uropathogenic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes food-borne disease in humans ranging from watery diarrhea to bloody diarrhea and severe hemorrhagic colitis, renal failure and hemolytic uremic syndrome. Cattle, the most important source of E. coli O157:H7 transmission to humans, harbor the bacteria in their gastrointestinal tract without showing clinical symptoms. Prevention of E. coli O157:H7 infections in ruminants could diminish the public health risk. However, there is no specific treatment available nor a vaccine or a therapeutic agent which completely prevents E. coli O157:H7 infections in cattle. This paper provides an overview of latest research data on eradicating enterohemorrhagic E. coli O157:H7 in ruminants by use of bovine lactoferrin administration. The article provides insights into the anti-microbial and immunomodulatory activities of bovine lactoferrin against E. coli O157:H7 infections in cattle.

Topics: Animals; Anti-Bacterial Agents; Cattle; Escherichia coli Infections; Escherichia coli O157; Feces; Humans; Lactoferrin

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.

Topics: Animals; Antibodies, Bacterial; Antibodies, Neutralizing; Antibody Specificity; Cattle; Cattle Diseases; Cell Line, Tumor; Colon; Escherichia coli Infections; Escherichia coli O157; Female; Hemolytic-Uremic Syndrome; Humans; Immunization; Immunoglobulin G; Lactoferrin; Male; Mice; Neutralization Tests; Pregnancy; Rats; Shiga Toxin 2

Topics: Animals; Anti-Infective Agents; Bacterial Vaccines; Cattle; Cattle Diseases; Disease Models, Animal; Enteropathogenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae, Bacterial; Genetic Predisposition to Disease; Hemolysin Proteins; Humans; Lactoferrin; Mice; Phagocytes; Swine; Swine Diseases; Vaccines, Attenuated

Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against

Topics: Anti-Infective Agents; Escherichia coli; Escherichia coli Infections; Humans; Lactoferrin; Microbial Sensitivity Tests; Peptides; Staphylococcal Infections; Staphylococcus aureus

Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 μmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.

Topics: Anti-Bacterial Agents; Cell-Penetrating Peptides; Escherichia coli; Escherichia coli Infections; Humans; Lactoferrin

LF-6 is a modified antibacterial peptide derived from LFP-20, a major active ingredient of porcine lactoferrin, whose antibacterial activity is 200 times higher than its native protein counterpart. Moreover, LF-6 displays even higher antibacterial activity than LFP-20 and negligible toxic adverse effects, make it a potential therapeutic agent for antibacterial purposes. Escherichia coli expression system has been a preferred choice and workhorse for most recombinant proteins. However, LF-6 must be coexpressed with a fusion partner to avoid its potentially fatal toxicity which would threat E. coli expression system. In this study, we successfully introduced intein system to solve this problem, which LF-6 was N-terminally fused to dithiothreitol (DTT)-induced self-cleavable intein, and it conduct cleavage when the intein-fusion peptide passing through a chromatography column filled with chitin, then the spliced peptide was purified with RP-HPLC and identified with mass spectroscopy. A bacteriostatic test showed that the recombinant LF-6 displayed nearly the same antibacterial activity as the chemically synthetized LF-6, and an in vivo immunoprotection analysis showed that the recombinant LF-6 exerted protective effects on Escherichia coli (ETEC)-K88-infected mice, which significantly reduced the pro-inflammatory cytokines level in plasma and intestine, and resistant to intestinal mucosal injury compared to the infective alone groups. Our study indicates that the intein system allows a safe and efficient method to produce recombinant LF-6, which not only has antibacterial activity, but more importantly, has an immunomodulatory function.

Topics: Animals; Anti-Infective Agents; Cytokines; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Feasibility Studies; Immunologic Factors; Inflammation Mediators; Inteins; Intestinal Mucosa; Lactoferrin; Male; Mice; Mice, Inbred ICR; Peptide Fragments; Recombinant Fusion Proteins; Swine

Enhancement of microbial biofilm formation by low antimicrobial doses is a critical problem in the medical field. The objective of this study was to propose a new drug candidate against the biofilm formation promoted by subinhibitory dose of antimicrobials. To determine the effect on biofilm formation of Escherichia coli, a subinhibitory concentration of lactoferrin (LF), a milk protein involved in a broad range of biological properties including antimicrobial action, or ampicillin (AMP), a typical antibiotic, was added to an E. coli cell culture in a 96-well microtiter plate. On the other hand, warfarin (WARF), an oral anticoagulant, or polymyxin B (PMB), a strong antibiotic for biofilm treatment, was added as an antagonist against the biofilm promoted by LF or AMP. The amount of biofilm formed at 100μg/mL LF in lysogeny broth medium was four times higher than in the absence of LF. Meanwhile, it was found that WARF suppressed the LF-promoted biofilm formation to a level comparable with the LF-free condition. WARF worked in a similar manner to PMB, which is known as an antibiofilm agent. Furthermore, WARF could also suppress the biofilm promoted by AMP. In conclusion, this study suggests that WARF can work as an antibiofilm agent against the biofilm formation promoted by subinhibitory dose of antimicrobials.

Topics: Ampicillin; Anti-Bacterial Agents; Biofilms; Escherichia coli; Escherichia coli Infections; Lactoferrin; Microbial Sensitivity Tests; Warfarin

Enterohemorrhagic Escherichia coli (EHEC) strains, of which E. coli O157:H7 is the best-studied serotype, are an important group of foodborne pathogens causing severe illness in humans worldwide. The main reservoirs for EHEC are ruminants, mostly cattle, which harbor the bacteria in their intestinal tracts without showing clinical symptoms. In this study, we used bovine lactoferrin, a natural occurring bactericidal and immunomodulating protein, as an antibacterial agent against EHEC infection in cattle. Nine 3-month-old Holstein-Friesian calves were experimentally infected with EHEC (strain NCTC12900). Three animals received a daily rectal spray treatment with bovine lactoferrin, three animals received an oral treatment, and three animals served as a control group. Blood samples were collected weekly and fecal samples twice weekly to monitor antibody responses and fecal excretion, respectively. Animals in the rectal group ceased shedding within 26 days of the experimental treatment and remained negative. This beneficial effect of bovine lactoferrin was not observed in the oral group, where animals were still shedding at the time of euthanasia (day 61). All groups developed serum responses, but no clear differences could be observed between the groups. However, the results indicate that the use of bovine lactoferrin as a rectal treatment can be a useful strategy to preclude further transmission of EHEC infections from cattle to humans.

Topics: Administration, Rectal; Animals; Anti-Bacterial Agents; Bacterial Shedding; Cattle; Disease Transmission, Infectious; Escherichia coli Infections; Escherichia coli O157; Feces; Lactoferrin; Treatment Outcome

Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to the surface charges. Analysis of Lfcin B-induced shape changes indicated that the binding of Lfcin B increased the area of the outer monolayer of GUVs. These results indicate that Lfcin B-induced damage of the plasma membrane of E. coli with its concomitant rapid leakage of internal contents is a key factor for the bactericidal activity of LfcinB.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Cattle; Cell Membrane Permeability; Escherichia coli; Escherichia coli Infections; Fluoresceins; Fluorescent Dyes; Humans; Lactoferrin; Molecular Sequence Data; Organic Chemicals; Phosphatidylcholines; Phosphatidylglycerols; Static Electricity; Sucrose; Unilamellar Liposomes

Crohn's disease is a multifactorial disease in which an aberrant immune response to commensal intestinal microbiota leads to chronic inflammation. The small intestine of patients with Crohn's disease is colonized by a group of adherent-invasive Escherichia coli strongly able to adhere and invade intestinal epithelial cells lactoferrin is an iron-binding glycoprotein known to have anti-bacterial and anti-inflammatory activities.. We explore the ability of bovine lactoferrin to modulate the interactions between the adherent-invasive E. coli strain LF82 and intestinal epithelial cells as well as the inflammatory response.. Bacterial adhesion and invasion assays were used to assess the antimicrobial activity of lactoferrin. Electron microscopy was used to characterize bacteria-cell interactions. The mRNA expression of pro-inflammatory cytokines was measured both in cultured cells and in biopsies taken from intestine of patients affected by Crohn's disease.. Lactoferrin inhibited bacterial invasion through minimally affecting adhesion. This divergence was due to a mannose-dependent lactoferrin binding to the bacterial type 1 pili and consequent bacterial aggregation on the intestinal epithelial cell surface. Expression of pro-inflammatory cytokines, such as TNF-alpha, IL-8, and IL-6, was markedly inhibited by lactoferrin both in cultured and Crohn-derived intestinal cells.. Bovine lactoferrin might function via an antibacterial and/or anti-inflammatory mechanism in the treatment of Crohn's disease.

Topics: Animals; Anti-Infective Agents; Bacterial Adhesion; Caco-2 Cells; Cattle; Crohn Disease; Escherichia coli; Escherichia coli Infections; Gene Expression; Humans; Interferon-gamma; Interleukin-6; Interleukin-8; Intestinal Mucosa; Lactoferrin; Mannose; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Receptors, Immunologic; RNA, Messenger; Tumor Necrosis Factor-alpha

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteremia; Cattle; Drug Therapy, Combination; Escherichia coli Infections; Escherichia coli O157; Feces; Intestines; Kanamycin; Kidney; Lactoferrin; Liver; Male; Mice; Mice, Inbred BALB C

Ruminants are an important reservoir of Escherichia coli O157:H7, therefore reducing E. coli O157:H7 excretion by these animals could play a key role in reducing human infections. The present study investigates the potential of bovine lactoferrin, a natural antimicrobial-immunomodulatory protein of milk, to prevent colonization and excretion of E. coli O157:H7 in sheep. The effect of two different doses of lactoferrin (1.5 g or 0.15 g per 12h) was evaluated on colonization of sheep intestine and faecal excretion of the NCTC12900 strain. Hereto, lactoferrin was orally administered to sheep during 30 consecutive days and sheep were experimentally infected with E. coli O157:H7 on the second day of the lactoferrin administration. Interestingly, both lactoferrin dosages significantly reduced the number of E. coli O157:H7 in faeces as well as the duration of faecal excretion. The high dose group showed a significantly higher antibody response against EspA and EspB, two structural proteins of the bacterial type III secretion system (TTSS), than the colonization control group. The results suggest that oral lactoferrin administration could be used to prevent persistent colonization of sheep with E. coli O157:H7.

Topics: Administration, Oral; Animals; Antigens, Bacterial; Bacterial Secretion Systems; Cattle; Escherichia coli Infections; Escherichia coli O157; Feces; Humans; Immunoglobulin G; Intestines; Lactoferrin; Sheep; Sheep Diseases; Sheep, Domestic

Diarrhea is a major public health problem that affects the development of children. Anthropometric data were collected from 274 children with (N = 170) and without (N = 104) diarrhea. Stool specimens were analyzed by conventional culture, polymerase chain reaction for enteroaggregative Escherichia coli (EAEC), Shigella, Cryptosporidium, Entamoeba, and Giardia species, and by enzyme-linked immunosorbent assay for fecal lactoferrin levels. About 50% of the study population was mildly to severely malnourished. Fecal lactoferrin levels were higher in children with diarrhea (P = 0.019). Children who had EAEC infection, with or without diarrhea, had high mean lactoferrin levels regardless of nutritional status. The EAEC and Cryptosporidium were associated with diarrhea (P = 0.048 and 0.011, respectively), and malnourished children who had diarrhea were often co-infected with both Cryptosporidium and EAEC. In conclusion, the use of DNA-biomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated lactoferrin was associated with diarrhea in this group of children.

Topics: Case-Control Studies; Child Nutrition Disorders; Child, Preschool; Cross-Sectional Studies; Diarrhea; Dysentery, Bacillary; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Feces; Female; Gastroenteritis; Ghana; Humans; Infant; Infant, Newborn; Lactoferrin; Male; Parasitic Diseases; Polymerase Chain Reaction; Prospective Studies; Virulence

Lactoferrin has antimicrobial activity associated with peptide fragments lactoferricin (LFC) and lactoferrampin (LFA) released on digestion. These two fragments have been expressed in Photorhabdus luminescens as a fusion peptide linked to protein cipB. The construct cipB-LFC-LFA was tested as an alternative to antimicrobial growth promoters in pig production. Sixty piglets with an average live body weight of 5.42 (sem 0.59) kg were challenged with enterotoxigenic Escherichia coli and randomly assigned to four treatment groups fed a maize-soyabean meal diet containing either no addition (C), cipB at 100 mg/kg (C+B), cipB-LFC-LFA at 100 mg/kg (C+L) or colistin sulfate at 100 mg/kg (C+CS) for 3 weeks. Compared with C, dietary supplementation with C+L for 3 weeks increased daily weight gain by 21 %, increased recovery from diarrhoea, enhanced serum glutathione peroxidase (GPx), peroxidase (POD) and total antioxidant content (T-AOC), liver GPx, POD, superoxide dismutase and T-AOC, Fe, total Fe-binding capacity, IgA, IgG and IgM levels (P < 0.05), decreased the concentration of E. coli in the ileum, caecum and colon (P < 0.05), increased the concentration of lactobacilli and bifidobacteria in the ileum, caecum and colon (P < 0.05), and promoted development of the villus-crypt architecture of the small intestine. Growth performance was similar between C+L- and C+CS-supplemented pigs. The present results indicate that LFC-LFA is an effective alternative to the feed antibiotic CS for enhancing growth performance in piglets weaned at age 21 d.

Topics: Animal Feed; Animals; Anti-Bacterial Agents; Antioxidants; Bacterial Proteins; Cattle; Colistin; Diarrhea; Dietary Supplements; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Genetic Engineering; Intestinal Mucosa; Lactoferrin; Lactoglobulins; Liver; Male; Peptide Fragments; Random Allocation; Recombinant Proteins; Swine; Swine Diseases; Weaning

Nosocomial infection with antibiotic-resistant strains is a major threat to critical care medicine. Selective decontamination of the digestive tract (SDD) is one of the strategies used to reduce ventilator-associated pneumonia and sepsis in critically ill patients. In the present study, we performed pathogenic challenges of the digestive tract in a transgenic milk-fed animal model to test whether porcine lactoferrin (pLF) is an effective SDD regimen.. Transgenic mice expressing recombinant pLF in their milk at a mean+/-SD concentration of 120+/-13.6 mg/L during the lactation stage fed normal CD-1 mice pups for 4 weeks. The pups were subsequently challenged with pathogenic Escherichia coli, Staphylococcus aureus, and Candida albicans.. Compared with the control groups fed wild-type (normal) milk, the groups fed pLF-enriched milk demonstrated statistically significant improvements in weight gain; lower bacterial numbers in intestinal fluid, blood, and liver; healthier microvilli in the small intestine; and alveoli in the lungs.. Our results showed that oral administration of pLF-enriched milk to mice led to broad-spectrum antimicrobial activity in the digestive tract and protected the mucosa of the small intestine from injury, implying that pLF can be used as an effective SDD regimen.

Topics: Animals; Animals, Newborn; Bacteremia; Bacterial Infections; Body Weight; Candidiasis; Cytokines; Escherichia coli Infections; Gastrointestinal Tract; Immunohistochemistry; Intestines; Lactation; Lactoferrin; Lung; Mice; Mice, Transgenic; Milk; Polymerase Chain Reaction; Recombinant Proteins; Staphylococcal Infections; Swine

Topics: Anti-Bacterial Agents; Drug Resistance, Bacterial; Drug Synergism; Escherichia coli; Escherichia coli Infections; Fluoroquinolones; Lactoferrin; Microbial Sensitivity Tests; Reverse Transcriptase Polymerase Chain Reaction; Urinary Tract Infections

Both lactoferrin (LF) and bacteriophages are potent antibacterial agents. LF is contained in the secretory fluids of mammals and bacteriophages are specific bacterial viruses.. The aim of this investigation was to determine whether combined treatment of infected mice may allow lowering the therapeutic dose of specific bacteriophages for Escherichia coli and Staphylococcus aureus.. CBA mice were infected intravenously (i.v.) with sublethal doses of E. coli or S. aureus and the specific T4 or A5 bacteriophages, respectively, were administered intraperitoneally (i.p.) or per os one hour following infection. The numbers of colony-forming units (CFUs) were determined in the livers after 24 hours.. Comparative administration of bacteriophages i.p. or per os showed that both routes of administration were equally efficacious in the protective action of bacteriophages. The bacteriophages were still very potent in reducing CFU numbers in the liver at a dose of 10(5)/mouse. Application of bovine lactoferrin (LF), 10 mg i.v., 24 h before infection, was also very effective in reducing CFU numbers. Using suboptimal (10(3)-10(4)) doses of bacteriophages and administration of LF, a more potent protective effect in reducing the CFU numbers in the infected mice was demonstrated. The combined effect of LF and bacteriophages in reducing CFU numbers was significantly higher than the effects of either agent alone. The study demonstrated that the combined application of LF and bacteriophages can significantly lower (1000 times) the effective dose of bacteriophages in reducing CFU numbers in infected mice.

Topics: Administration, Oral; Animals; Bacteriophage T4; Cattle; Colony Count, Microbial; Dose-Response Relationship, Drug; Escherichia coli; Escherichia coli Infections; Female; Injections, Intraperitoneal; Injections, Intravenous; Lactoferrin; Liver; Male; Mice; Mice, Inbred CBA; Staphylococcal Infections; Staphylococcus; Staphylococcus Phages

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

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

Lactoferrin, an iron-binding glycoprotein found in cervical mucus and amniotic fluid, plays a defensive role against mucosal infections. This study examined the effect of recombinant human lactoferrin on preterm delivery in a rabbit model.. Anesthetized rabbits were randomly assigned to receive either inoculation with Escherichia coli or saline solution and to receive treatment with or without recombinant human lactoferrin inserted into the cervix 2 hours before bacterial inoculation (condition A: saline + saline; condition B: E coli + saline; condition C: E coli + recombinant human lactoferrin). E coli , saline solution, and recombinant human lactoferrin were inserted into the cervix using a hysteroscope and a sterile polyethylene cannula. Fetus survival rate and days to delivery after inoculation were monitored and tumor necrosis factor-alpha concentrations were measured in maternal serum and amniotic fluid.. Fetus survival for conditions A, B, and C were 95.7%, 0%, and 32.6%, respectively, whereas pregnancy continuation was 7.00 +/- 0 days, 3.25 +/- 0.43 days, and 4.85 +/- 1.77 days, respectively.. Cervical recombinant human lactoferrin administration increased fetal survival and extended pregnancy. Lactoferrin has an anti-inflammatory action as well as an antibacterial action, suggesting that recombinant human lactoferrin has the potential to prevent preterm delivery originating from cervical infection in the clinical setting.

Topics: Animals; Disease Models, Animal; Escherichia coli Infections; Female; Fetal Death; Fetal Membranes, Premature Rupture; Humans; Immunohistochemistry; Lactoferrin; Obstetric Labor, Premature; Placenta; Pregnancy; Pregnancy, Animal; Primary Prevention; Rabbits; Random Allocation; Recombinant Proteins; Reference Values; Sensitivity and Specificity; Tumor Necrosis Factor-alpha

Mechanisms for protecting spermatozoa, and the testes that produce them, from infection are essential, given the importance of these cells and organs for the fertility of the individual and perpetuation of the species. This is borne out by the publication of numerous papers on this subject over the last 50 years. We extended our work and that of others on the anti-infectious defense system of the male genital tract, using a new strategy for the direct identification of antibacterial molecules in human seminal plasma. We subjected a liquefied seminal plasma cationic fraction to reversed-phase HPLC, monitored microbicidal activity by gel overlay and radial diffusion assays, and identified the proteins and/or peptides present in each active fraction by mass spectrometry. In addition to proteins with known potent microbicidal activity--phospholipase A2, lactoferrin, and lysozyme--we also found that peptides produced by cleavage of semenogelin I, the predominant human semen coagulum protein, had high levels of antibacterial activity.

Topics: Amino Acid Sequence; Cations; Cell Fractionation; Chromatography; Chromatography, High Pressure Liquid; Electrophoresis; Escherichia coli Infections; Humans; Lactoferrin; Male; Molecular Sequence Data; Muramidase; Peptide Fragments; Phospholipases A; Phospholipases A2; Semen; Seminal Vesicle Secretory Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Previous studies demonstrated that lactoferrin (LF), given intravenously (i.v.), 24 h before lethal Escherichia coli ( E. coli) infection, protects mice against mortality. The aim of this investigation was to determine whether downregulation of serum TNF alpha activity and increase of neutrophil number in the circulation and bone marrow by LF could contribute to the protective action of LF against E. coli-induced sepsis.. CBA female mice, 10-12 week old, weight 20-22 g, were used.. Mice were given 10 mg LF i.v. either 2 h or 24 h before i.v. administration of lethal dose of E. coli (5 x 10(8)).. Serum activities of TNF alpha and IL-1 were determined by bioassays 2 h following E. coli or LF injection. The blood and bone marrow smears were stained with Giemsa and May-Grünwald reagents and reviewed histologically.. LF given 24 h before E. coli caused a 60% reduction of TNF alpha released into circulation. However, pretreatment of mice with LF 2 h before bacterial challenge resulted in strong (15 fold) increase of TNF alpha serum level. Analysis of bone marrow cell composition revealed a significant increase in neutrophil lineage cell content (myelocytes, bands and mature neutrophils) following 24 h pretreatment with LF (51.8% of the total cell count), versus PBS control (32.7%) and 2 h LF pretreatment (35.8%). The percentage of neutrophils (bands and mature forms) in the peripheral blood rose to 47.4% versus 32% and 32%, respectively. Intravenous administration of LF increased also interleukin 1 (IL-1) concentration in the circulation of noninfected mice.. This investigation has added more information regarding the mechanism of the protective action of LF in E. coli-induced bacteremia by revealing the phenomenon of accelerated neutrophil recruitment and down-regulation of E. coli-induced TNF alpha serum level.

Topics: Animals; Bacteremia; Bone Marrow; Dose-Response Relationship, Drug; Escherichia coli; Escherichia coli Infections; Female; Interleukin-1; Lactoferrin; Mice; Mice, Inbred CBA; Neutrophils; Sepsis; Time Factors; Tumor Necrosis Factor-alpha

Previous studies on cyclophosphamide (CP)-immunocompromised mice showed accelerated reconstitution of immune system function following oral treatment with lactoferrin (LF). The aim of this investigation was to evaluate the ability of mice, treated with a sublethal dose of CP and given LF, to combat bacterial infections. Mice were injected with a single, intraperitoneal dose of CP (350 mg/kg body weight). One group of CP-treated mice was also given LF in drinking water (0.5% solution) for 14 days. Untreated and LF-treated mice served as controls. On day 15 following CP administration, mice were infected intravenously with 10(8) Escherichia coli or 5 x 10(7) Staphylococcus aureus. Twenty-four hours later, the number of colony-forming units (CFU) in spleens and livers were determined. Phenotypic analysis of blood leukocytes was determined, as well as the ability of splenic and peritoneal cells to produce IL-6 spontaneously and in the presence of lipopolysaccharide (LPS). Treatment with CP, or with CP and LF, led to profound reduction of E. coli CFU in the liver and the spleen; treatment with LF alone had significant inhibitory effects on organ enumerated CFU. S. aureus CFUs were also significantly reduced in spleens of mice treated with CP or CP/LF and, to a lesser degree, after LF alone. These effects were also significantly reduced in the livers. Analysis of blood cellular phenotype revealed total number of peripheral leukocytes was lower in the CP-treated group (52.6%) but not significantly different from control values in CP/LF and LF-treated groups (90.7% and 104.6%, respectively). Conversely, percentage of blood neutrophils was markedly elevated in CP and CP/LF groups--62% and 42.5% vs. 18.4% in controls. These findings were accompanied by production of IL-6 by splenic and peritoneal cells which was significantly increased in CP- and CP/LF-treated groups. It was concluded that the increased clearance of bacteria in the organs of mice treated with CP and CP/LF may result from a rise in the number of neutrophils infiltrating the organs and contributing to accelerated clearance of bacteria. The study also suggests that the ability of cells from CP- and CP/LF-treated mice to produce significantly more IL-6 may also contribute to increased resistance to infections. Lastly, together with our previous data, this study indicates that LF used to reconstitute the antigen-specific immune response in CP-treated mice does not impair their resistance to infection.

Topics: Animals; Cyclophosphamide; Escherichia coli Infections; Female; Immunosuppressive Agents; Interleukin-6; Lactoferrin; Leukocyte Count; Leukocytes; Lipopolysaccharides; Liver; Macrophages, Peritoneal; Male; Mice; Mice, Inbred CBA; Spleen; Staphylococcal Infections

C3H/HeCr mice are more susceptible to infection compared with other strains. Lactoferrin (LF), a protein involved in innate defense, was shown to protect mice against lethal endotoxemia. In this investigation we attempt to explain the cause of increased susceptibility of C3H/HeCr mice to LPS and lack of protective LF action in these mice. We found that C3H/HeCr mice produced up to 5-fold more serum TNFalpha and 66% higher IFNgamma levels in response to i.v. LPS injection than the control, CBA strain. 24 h pretreatment of C3H/HeCr mice with LF did not cause inhibition of the LPS-induced TNFalpha serum levels, whereas in CBA mice LF significantly decreased TNFalpha level. IL-6 serum levels, in turn, were lowered in C3H/HeCr mice but elevated in CBA mice. That differential regulation of cytokine production by LF in C3H/HeCr mice paralleled a decreased survival after lethal LPS injection - 10% vs. 60% in control, PBS treated mice. In addition, determination of colony forming units (CFU) in livers and spleens after administration of 10(8) Escherichia coli revealed that pretreatment of CBA mice with LF caused a marked reduction of CFU in these organs, whereas in C3H/HeCr mice the changes were insignificant. These results indicate that the altered TNFalpha/IL-6 ratio in C3H/HeCr mice, as compared to control CBA mice, as well as the increased IFNgamma level, may be responsible for the increased susceptibility to endotoxemia in that substrain. We also suggest that an association exists between the LF protective effect against endotoxic sequelae and the insult-induced systemic immune response.

Topics: Animals; Cytokines; Endotoxemia; Escherichia coli; Escherichia coli Infections; Interferon-gamma; Interleukin-6; Lactoferrin; Lipopolysaccharides; Mice; Mice, Inbred C3H; Mice, Inbred CBA; Tumor Necrosis Factor-alpha

Topics: Bacterial Adhesion; Bacterial Outer Membrane Proteins; Cells, Cultured; Diarrhea, Infantile; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Humans; Infant; Lactoferrin

Faecal lactoferrin, an iron-based glycoprotein found concentrated in secondary granules of neutrophils, may serve as a surrogate marker of inflammation in the intestine. We evaluated the usefulness of faecal lactoferrin as a predictor of infection with invasive enteropathogens in 262 children with diarrhoea. Lactoferrin at a dilution of 1:50 had the highest sensitivity for detection not only of conventionally cultured invasive enteropathogens but also of all other enteropathogens. Neither individual clinical symptoms nor the identification of faecal leucocytes by microscopy significantly predicted isolation of invasive enteropathogens from the faeces of children with diarrhoea. Faecal lactoferrin is a simple test which showed promise in predicting which children with diarrhoea are likely to be infected with invasive pathogens and can be incorporated as a screening test before faecal cultures are undertaken in this population.

Topics: Acute Disease; Bacterial Infections; Biomarkers; Child; Child, Preschool; Diarrhea; Escherichia coli Infections; Feces; Female; Humans; India; Infant; Lactoferrin; Leukocytes; Male; Predictive Value of Tests; Sensitivity and Specificity

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. EPEC uses a type III secretory system to deliver effector proteins into the host cell. These proteins cause the characteristic attaching and effacing lesion on enterocytes. Lactoferrin, a glycoprotein present in human milk, inhibits EPEC adherence to mammalian cells. To determine the effect of lactoferrin on the initial host cell attachment step that is mediated by the type III secretory system, we focused on EPEC-induced actin polymerization in HEp2 cells, on the hemolytic activity, and on measurement of E. coli secreted proteins A, B, and D (EspABD). Lactoferrin blocked EPEC-mediated actin polymerization in HEp2 cells and blocked EPEC-induced hemolysis. The mechanism of this inhibition was lactoferrin-mediated degradation of secreted proteins necessary for bacterial contact and pore formation, particularly EspB. The proteolytic effect of lactoferrin was prevented by serine protease inhibitors. This disruption of the type III secretory system implies that lactoferrin could provide broad cross protection against the enteropathogens that share this mechanism.

Topics: Actins; Bacterial Adhesion; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cell Line; Diarrhea; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Hemolysis; Humans; In Vitro Techniques; Infant; Lactoferrin; Recombinant Proteins

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

Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.

Topics: Animals; Animals, Newborn; Cell Death; Colony Count, Microbial; DNA-Binding Proteins; Drug Combinations; Early Growth Response Protein 1; Escherichia coli; Escherichia coli Infections; Female; Humans; Immediate-Early Proteins; Intestines; Lactoferrin; Liver; Macrophages; Muramidase; NF-kappa B; Nitric Oxide; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Severity of Illness Index; Transcription Factors; Tumor Necrosis Factor-alpha

We have previously shown that bovine lactoferrin (BLF) given intravenously (i.v.) protected mice against a lethal dose of Escherichia coli and strongly stimulated both the clearing and killing activities in liver, lungs, spleen and kidney. Since some studies indicated a reduction of the manifestation of experimental pancreatitis with lactoferrin (LF), we decided to examine the protective activity of BLF against lethal E. coli infection in animals with alloxan (Alx)-induced diabetes. It appeared that 48 h diabetes substantially lowered the killing activity in all four organs as well as the clearing rate of E. coli from the circulation. BLF given i.v. reduced this undesirable effect of diabetes. However, in 10- and 20-day diabetic animals, the diabetes alone stimulated the killing activity in the organs investigated, and upregulated the clearing rate of E. coli from the circulation. Lactoferrin significantly increased both the killing and the clearing activity in these long-term diabetic animals. In some cases the stimulating effect of BLF was very high, suggesting a concerted action of BLF and diabetes in that category of mice. Despite these beneficial effects of BLF and diabetes on the killing process in the investigated organs, the survival time of animals from all the diabetic groups (48 h, 10 and 20 days) was not prolonged by BLF. The protective properties of BLF did not depend on the blood glucose levels in the diabetic animals. BLF partly delayed the development of experimental Alx-induced diabetes, measured by the glucose level, but only if administered shortly after Alx injection. In conclusion, we demonstrated that the state of diabetes alone could increase killing of bacteria in the investigated organs and LF enhanced this process. However, LF had no protective effect against the mortality of diabetic mice infected with a lethal dose of E. coli.

Topics: Animals; Blood Glucose; Cattle; Diabetes Mellitus, Experimental; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Male; Mice; Survival Rate

Lactoferrin (LF) is a multifunctional immunoregulatory protein that has been associated with host defense at mucosal surfaces through its antibacterial properties. The antibacterial and anti-inflammatory properties of LF were further explored with an animal model of experimental urinary tract infection. Bovine LF (bLF), human LF (hLF), and synthetic peptide sequences based on the antibacterial region of hLF (amino acid residues 16 to 40 [HLD1] and 18 to 40 [HLD2]) were given orally to female mice 30 min after the instillation of 10(8) Escherichia coli bacteria into the urinary bladder. The control groups received phosphate-buffered saline or water. C3H/Tif mice were treated with hLF or bLF, and C3H/HeN mice were treated with bLF only. The numbers of bacteria in the kidneys and bladder of C3H/Tif and C3H/HeN mice were significantly reduced 24 h later by the LF treatments compared to the findings for the control group. The hLF-treated group showed the strongest reduction compared with the vehicle-treated-group (P values were 0.009 and 0.0001 for the kidneys and bladder, respectively). The urinary leukocyte response was diminished in the hLF-treated group. The hLF treatment also significantly reduced the urinary interleukin-6 (IL-6) levels at 2 h and the systemic IL-6 levels at 24 h after infection (P values were 0.04 and < 0.002, respectively). In the bLF-treated animals, no such strong anti-inflammatory effects were obtained. In another series of experiments, C3H/Tif mice perorally treated with HLD1 or HLD2 also showed reduced numbers of bacteria in the kidneys compared with the vehicle-treated mice, although the results were significantly different only for HLD2 (P < 0.01). Analysis of urine from hLF-fed C3H/Tif mice showed that hLF was excreted into the urinary tract at 2 h after feeding. Testing of the in vitro bactericidal activity of LF (1 mg/ml) or the peptides (0.1 mg/ml) in mouse urine against the E. coli bacteria revealed moderate killing only by HLD2. In conclusion, these results demonstrate for the first time that oral administration of hLF or peptides thereof is effective in reducing infection and inflammation at a remote site, the urinary tract, possibly through transfer of hLF or its peptides to the site of infection via renal secretion. The antibacterial mechanism is suggested to involve bactericidal capacities of LF, fragments thereof, or its peptides.

Topics: Amino Acid Sequence; Animals; Bacterial Adhesion; Cattle; Child; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Female; Humans; Interleukin-6; Kidney; Lactoferrin; Mice; Mice, Inbred C3H; Molecular Sequence Data; Peptides; Urinary Tract Infections

As part of a traveler's diarrhea study carried out in Guadalajara, Mexico, and Goa, India, we conducted a case control study to evaluate fecal markers of enteric inflammation in three groups. Forty-five cases of enteroaggregative Escherichia coli (EAEC) diarrhea were compared to 56 controls with enterotoxigenic E. coli (ETEC) diarrhea, and 126 controls with diarrhea without identifiable pathogens. For EAEC cases we found fecal leukocytes, occult blood, and lactoferrin in 13 (28.9%), 14 (31.1%), and 27 (60.0%) patients, respectively; for ETEC controls they were 15 (26.8%), 16 (28.6%), and 15 (26.8%) respectively; and for patients without identifiable pathogens 19 (15.1%), 34 (27.0%) and 27 (21.4%) were seen for the presence of a positive fecal lactoferrin test in EAEC cases was statistically significant compared to both control groups. The study provides evidence that EAEC infection is associated with an intestinal inflammatory response.

Topics: Adolescent; Adult; Bacterial Adhesion; Biomarkers; Case-Control Studies; Cell Line; Diarrhea; Enteritis; Escherichia coli; Escherichia coli Infections; Feces; Humans; Lactoferrin; Leukocytes; Occult Blood; Travel

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

Enteroaggregative E. coli (EAggEC) are emerging as an important cause of persistent diarrhea, especially in children in the developing world, yet the pathogenesis of EAggEC infection is poorly understood. In an ongoing prospective study of childhood diarrhea in an urban Brazilian slum, EAggEC are the leading cause of persistent diarrhea. Children from this study with EAggEC and persistent diarrhea had significant elevations in fecal lactoferrin, interleukin (IL)-8, and IL-1beta. Moreover, children with EAggEC without diarrhea had elevated fecal lactoferrin and IL-1beta concentrations. The children with EAggEC in their stool had significant growth impairment after their positive culture, regardless of the presence or absence of diarrhea. Finally, 2 EAggEC strains were shown to cause IL-8 release from Caco-2 cells, apparently via a novel heat-stable, high-molecular-weight protein. These findings suggest that EAggEC may contribute to childhood malnutrition, trigger intestinal inflammation in vivo, and induce IL-8 secretion in vitro.

Topics: Brazil; Caco-2 Cells; Case-Control Studies; Cells, Cultured; Child, Preschool; Diarrhea; Escherichia coli; Escherichia coli Infections; Feces; Growth Disorders; Humans; Infant; Infant, Newborn; Inflammation; Interleukin-1; Interleukin-8; Intestines; Lactoferrin; Polymerase Chain Reaction; Prospective Studies; RNA, Messenger

A single dose of bovine lactoferrin (BLF) was given intravenously (i.v.) to CFW mice 24 hours (h) prior to the i.v. injection of the E. coli lethal dose (LD100). BLF strongly accelerated the clearance rate of E. coli from the blood as well as its killing rate in the liver, lungs, spleen and kidney. The highest clearing and killing rate was found 5 h after E. coli LD100 injection. The most intensive killing in the organs examined was found in the lungs and kidney. Analysis of organs of i.v. BLF-stimulated mice which survived up to day 30 after the infection by E. coli showed that not all animals were definitely pathogen-free. It was concluded that the defense system generated by BLF in mice in vivo is primarily a bacteria-killing one. The participation and cooperation of reticulo-endothelial (RE)-macrophages and granulocytes in the phagocytosis and killing of E. coli may thus be related to the protective activity of LF.

Topics: Animals; Blood Bactericidal Activity; Escherichia coli; Escherichia coli Infections; Female; Immunity, Innate; Injections, Intravenous; Kidney; Lactoferrin; Lung; Male; Mice; Mice, Inbred Strains; Time Factors

Topics: Colitis; Escherichia coli Infections; Feces; Gastrointestinal Hemorrhage; Humans; Lactoferrin

The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binding EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subepithelial matrix proteins and carbohydrates tested (in 10(4)-fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (Ka) of 1.4 x 10(-7) M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli.

Topics: Animals; Cattle; Escherichia coli; Escherichia coli Infections; Gastroenteritis; Humans; Lactoferrin

The clinical efficacy of a preparation based on the lactoperoxidase system (LP-s) and lactoferrin (LF) was tested in calves experimentally infected with E coli K99+, Ent+. Mortality, occurrence and duration of diarrhoea were significantly lower (P less than 0.05) and general clinical status significantly better (P less than 0.05) in infected calves treated with LP-s and LF preparation than in infected but non-treated calves. Results suggest that LP-s and lactoferrin are effective in the treatment of enteric colibacillosis in calves.

Topics: Animals; Body Weight; Cattle; Cattle Diseases; Diarrhea; Drug Therapy, Combination; Escherichia coli Infections; Feces; Glucose; Glucose Oxidase; Lactoferrin; Lactoglobulins; Lactoperoxidase; Peroxidases; Random Allocation; Thiocyanates

Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli. About 70% of mice pretreated with LB survived challenge. The survival rates in control mice treated with E. coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively. Human lactoferrin (LH) had almost the same protective effect as LB. Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E. coli administration. The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB. In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment. From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30. The possible mechanism(s) of protective effect of LB and role of iron ions are discussed.

Topics: Animals; Cattle; Chlorides; Drug Administration Schedule; Drug Interactions; Escherichia coli Infections; Female; Ferric Compounds; Iron; Lactoferrin; Lactoglobulins; Male; Mice; Mice, Inbred Strains

Total serum iron, plasma lactoferrin and circulating leukocytes were measured in piglets during the early phase of severe gram-negative septicemia and endotoxemia in 3 experimental settings: intravenous (i.v.) infusion of lipopolysaccharide (LPS) (n = 8), i.v. infusion of live Escherichia coli (n = 7) and intraperitoneal (i.p.) infusion of E. coli (n = 6). Iron dropped significantly during the first 30 min of LPS infusion from a median of 32 microM to 13.4 microM. A similar decrease in serum iron was demonstrated in the 2 other groups with minimum values at 120 min after the start of E. coli infusion. Plasma levels of lactoferrin increased significantly 120 min after the start of LPS infusion (median 6 mg/l) when compared to preinfusion values (0.25 mg/l). After i.v. infusion of E. coli a significant rise of plasma lactoferrin was demonstrated already 30 min after bacterial infusion (to 2.1 mg/l) compared to preseptic values (0.8 mg/l). This increase was accompanied with a significant drop of circulating leukocytes (to 7.3 x 10(9)/l) compared to before the infusion (17 x 10(9)/l) in the pigs given E. coli i.v. After i.p. E. coli infusion no significant change of plasma lactoferrin was observed. The rapid fall of total serum iron seen during endotoxemia and E. coli septicemia may in part be explained by the release of lactoferrin from granulocytes and the clearance of iron-bound lactoferrin in the blood or peritoneal cavity.

Topics: Animals; Escherichia coli Infections; Female; Iron; Lactoferrin; Lactoglobulins; Leukocytes; Lipopolysaccharides; Male; Sepsis; Swine; Toxemia

The levels of plasma lactoferrin (LF) in response to endotoxin and Escherichia coli infusions in piglets were studied to obtain exact time relation of plasma LF increase in relation to start of endotoxin and E. coli infusions. A new enzyme-linked immunoassay of swine LF is presented. 13 piglets had a 10-fold rapid increase of plasma LF concentrations after 0.25 mg/kg endotoxin intravenous infusion. The initial rise was 3.4 mg/l/h. 14 piglets, receiving 1 x 10(11) E. coli intravenously, showed a higher increase of plasma LF concentrations, amounting to 6-9 mg/l/h. Thus, plasma LF was an early marker of septicemia and endotoxemia.

Topics: Anesthesia; Animals; Endotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Female; Halothane; Lactoferrin; Lactoglobulins; Leukocyte Count; Male; Sepsis; Swine; Toxemia

Mice with cyclophosphamide-induced granulocytopenia were challenged with aerosolized Escherichia coli, their lungs were lavaged at 1 and 4 h, and total cell counts, differential counts, and levels of lactoferrin, transferrin, and albumin were measured in the lung lavage fluid. Lung lavage fluid from cyclophosphamide-treated mice had few neutrophils and no increase in lactoferrin levels, whereas control mice had significant increases in both. Transferrin levels did not change in either group. Neutrophils are the source of increased lactoferrin levels in lung lavage fluid after aerosol challenge.

Topics: Aerosols; Albumins; Animals; Cyclophosphamide; Escherichia coli Infections; Lactoferrin; Lactoglobulins; Lung; Mice; Neutrophils; Transferrin

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

A total of 516 strains of Escherichia coli were screened for the presence and expression of the aerobactin iron uptake system. The incidence was markedly higher among clinical isolates from patients with septicemia (68.8%), pyelonephritis (74.6%), and symptomatic (59.8%) and asymptomatic (63.2%) lower urinary tract infections than among normal human fecal isolates (34.3%).

Topics: Bacteriuria; Cystitis; Escherichia coli; Escherichia coli Infections; Feces; Humans; Hydroxamic Acids; Iron; Lactoferrin; Pyelonephritis; Transferrin

Iron-binding proteins have antibacterial activity; they have been identified in lung secretions, but their role in pulmonary antibacterial defenses is unclear. Murine lactoferrin and murine transferrin were used to generate polyclonal antiserum to lactoferrin and to transferrin, and the specificity of both antisera was shown by western blot. Mice were exposed to either aerosolized Escherichia coli or Staphylococcus aureus; they were killed 1, 4, 24, or 48 hr later; and their lungs were lavaged. We measured the levels of transferrin, lactoferrin, and albumin and did a cell count for the lavage fluid. The predominant iron-binding protein in resting animals was transferrin. Aerosolized E. coli caused a brisk PMNL response in the lungs that was associated with a major increase in the levels of lactoferrin. Challenge with S. aureus was associated with a moderate increase in the number of macrophages and a moderate decrease in the levels of transferrin and iron but no change in the levels of lactoferrin. The levels of iron-binding protein can vary according to the type of inflammatory response.

Topics: Aerosols; Albumins; Animals; Escherichia coli Infections; Female; Iron; Lactoferrin; Lactoglobulins; Lung; Mice; Neutrophils; Staphylococcal Infections; Transferrin

The antibacterial activity of ovotransferrin (conalbumin) against different bacterial species was studied in vitro. The most sensitive species were Pseudomonas sp., E. coli, S. mutans; and the most resistant ones S. aureus, Proteus sp., Klebsiella. The bacteriostatic activity of conalbumin in various conditions was also tested. The presence of bicarbonate ions always increased the activity of conalbumin, while an antagonistic effect of citrate was observed in bacteria with a receptor for the iron-citrate complex. Experiments with conalbumin covalently linked to Sepharose 4B indicated that its antibacterial activity may not be due simply to the removal of iron from the medium, but probably involves other metals and an interaction with the bacterial surface. The in vitro studies carried out with conalbumin and lactoferrin demonstrated that the two proteins produced a similar inhibition of growth of E. coli and S. mutans. The in vivo studies showed that the protective effect of conalbumin in newborn guinea pigs with E. coli by gastrointestinal route was similar to that naturally provided by the lactoferrin present in milk.

Topics: Animals; Bacteria; Bicarbonates; Citrates; Citric Acid; Conalbumin; Egg Proteins; Escherichia coli Infections; Guinea Pigs; Hydrogen-Ion Concentration; Iron; Lactoferrin; Transferrin

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

In plasma clots the presence of ferritin-antiferritin complexes interferes with the bactericidal powers of polymorphs against Klebsiella pneumoniae and Escherichia coli. Equivalent concentrations of apoferritin-antiferritin complexes, which lack Fe, do not have this effect and it is therefore suggested that the iron-binding capacity of lactoferrin in polymorphs plays an essential role in the bactericidal power of the cell. Similar results were obtained in vivo where ferritin-antiferritin complexes cause a high mortality in otherwise non-lethal infections. Evidence suggests that it is the iron in the ferritin which is responsible for the rapid intracellular bacterial growth and that lactoferrin normally plays an important protective role within the polymorphs.

Topics: Animals; Antigen-Antibody Complex; Apoferritins; Blood Coagulation; Escherichia coli Infections; Ferritins; Klebsiella Infections; Klebsiella pneumoniae; Lactoferrin; Mice; Neutrophils; Rabbits

Previous work has shown that antibody and transferrin, acting together, exert a bacteriostatic effect on certain pathogenic Escherichia coli. This effect may be due to the ability of the antibody to interfere with the release of the iron chelator, enterochelin, from the bacterial cell. Enterochelin is essential for the transport of iron from transferrin to the bacterial cell. The nature of the bacterial antigen against which the antibody is directed has now been determined by means of adsorption experiments. It was found that absorption of serum either with hear-killed cells of E. coli O111 or with Boivin antigen abolished the bacteriostatic effect. A monosaccharide, which proved to be colitose (3,6-dideoxy-L-galactose), was isolated after acetic acid hydrolysis of the Boivin antigen. Colitose is the terminal monosaccharide of the O-specific side chain of the lipopolysaccharide from E. coli O111. This monosaccharide abolished the bacteriostatic effect of both whole serum and mixtures of antibody and iron-binding proteins. When administered by the intraperitoneal route, it reduced the resistance of mice to subsequent infection with E. coli O111. This ability of colitose to interfere with antibacterial mechanisms is in accord with published immunochemical studies.

Topics: Animals; Antibodies, Bacterial; Blood Physiological Phenomena; Deoxy Sugars; Escherichia coli; Escherichia coli Infections; Lactoferrin; Lipopolysaccharides; Mice; Transferrin

The bacteriostatic activity of guinea-pig milk against various strains of Escherichia coli has been examined. Milk collected from sows suckling normal young was usually inactive, but the activity of milk from sows suckling young which had been orally infected with Esch. coli was significantly increased. The increase occurred in 2 phases: the first was found as early as 24 h after infection, suggesting stimulation via the teat canal (diathelic), but lasted only 2-3 days; the second occurred from 10 days post-infection onwards, and lasted until the end of lactation. The occasional bacteriostatic activity of milk from sows with normal young was not correlated with the presence in the faeces of naturally occurring Enterobacteriaceae, including Esch. coli.

Topics: Animals; Antibodies, Bacterial; Bicarbonates; Escherichia coli; Escherichia coli Infections; Feces; Female; Guinea Pigs; Iron; Lactation; Lactoferrin; Milk; Pregnancy

Topics: Anemia, Hypochromic; Animals; Cattle; Escherichia coli Infections; Female; Food, Fortified; Gastroenteritis; Humans; Infant; Infant Nutritional Physiological Phenomena; Infant, Newborn; Infant, Premature, Diseases; Iron; Lactoferrin; Milk; Milk, Human; Protein Binding

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

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: Escherichia coli; Escherichia coli Infections; Humans; Infant Nutritional Physiological Phenomena; Infant, Newborn; Lactoferrin; Lactoglobulins; Protein Binding; Transferrin