Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.     

Detection of specific antibodies in cord blood, infant and maternal saliva and breast milk to staphylococcal toxins implicated in sudden infant death syndrome (SIDS)

The common bacterial toxins hypothesis of sudden infant death syndrome (SIDS) is that nasopharyngeal bacterial toxins can trigger events leading to death in infants with absent/low levels of antibody that can neutralise the toxins. The aim of this study was to investigate nasopharyngeal carriage of Staphylococcus aureus and determine levels of immunity in the first year of life to toxic shock syndrome toxin (TSST-1) and staphylococcal enterotoxin C (SEC). Both toxins have been implicated in SIDS cases. Seventy-three mothers and their infants (39 males and 34 females) were enrolled onto the study. The infants had birth dates spread evenly throughout the year. In infants, S. aureus carriage decreased significantly with age (P<0.001). Between 40% and 50% of infants were colonised with S. aureus in the first three months of life and 49% of the isolates produced one or both of the staphylococcal toxins. There was a significant correlation between nasopharyngeal carriage of S. aureus in mothers and infants in the three months following the birth (P<0.001). Carriage of S. aureus in infants and their mothers was not significantly associated with levels of antibody to TSST-1 or SEC in cord blood, adult saliva or breast milk. Infants colonised by S. aureus had higher levels of salivary IgA to TSST-1 than infants who were culture negative. Analysis of cord blood samples by a quantitative ELISA detected IgG bound to TSST-1 and SEC in 95.5% and 91.8% of cases respectively. There was a marked variation in levels of maternal IgG to both TSST-1 and SEC among cord blood samples. Maternal age, birth weight, and seasonality significantly affected the levels of IgG binding to TSST-1 or SEC. Analysis of infant saliva samples detected IgA to TSST-1 and SEC in the first month after birth; 11% of samples tested positive for salivary IgA to TSST-1 and 5% for salivary IgA to SEC. By the age of two months these proportions had increased to 36% and 33% respectively. More infants who used a dummy tested positive for salivary IgA to TSST-1 compared to infants who did not use a dummy. Levels of IgA to TSST-1 and SEC detected in the breast-milk samples varied greatly among mothers. There was a trend for infants receiving breast milk with low levels of antibody to TSST-1 or SEC to have higher levels of salivary antibody to the toxins. In conclusion, passive immunity to toxins implicated in SIDS cases varies greatly among infants. Infants are able to mount an active mucosal immune response to TSST-1 and SEC in the first month of life.     

Dietary intake of selenium and its concentration in breast milk

Selenium concentration was measured in the breast milk of 30 mothers at different stages of lactation and various body mass indices (BMI). For a maternal mean selenium intake meeting 100% of the Recommended Daily Allowance, mean milk selenium concentration was 14.06 ng/mL (range: 10.0–24.7 ng/mL). No significant correlation was found between the concentration of milk selenium with the stage of lactation, BMI, or dietary selenium intake.     

Antimicrobial potential of four Lactobacillus strains isolated from breast milk

AIMS: The antimicrobial potential of four lactobacilli (Lactobacillus salivarius CECT5713, Lactobacillus gasseri CECT5714, L. gasseri CECT5715 and Lactobacillus fermentum CECT5716), isolated from fresh human breast milk, was evaluated in this study and compared with Lactobacillus coryniformis CECT5711, a reuterin-producing strain isolated from an artisan goat's cheese. METHODS AND RESULTS: Agar diffusion tests, competitive adhesion assays and mucin expression assays were carried out in order to value the antibacterial properties of the lactobacilli strains. The antibacterial capability of the strains was tested in vivo by using a murine infection model with Salmonella choleraesuis. The results revealed that all the strains studied, displayed antibacterial properties against pathogenic bacteria. However, the antibacterial potential varied among the lactobacilli tested and, in fact, L. salivarius CECT5713 showed not only the best in vitro antibacterial activity, but also the highest protective effect against a Salmonella strain in the murine infection model. CONCLUSION: The four breast-milk lactobacilli, and particularly L. salivarius CECT5713, possess potent antibacterial activities that result in a higher protection against S. choleraesuis CECT4155 in a mouse infection model. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that lactobacilli from breast milk could contribute to an anti-infective protection in neonates and would be excellent candidates for the development of infant probiotic products.     

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

The role of microbicidal lipids in host defense against pathogens and their potential as therapeutic agents

Lipids such as fatty alcohols, free fatty acids and monoglycerides of fatty acids are known to be potent antimicrobial/microbicidal agents in vitro and to kill enveloped viruses, Gram-positive and Gram-negative bacteria and fungi on contact. For over half a century several studies have tried to answer the question of whether or not lipids play a role in the natural host defense against pathogens. A comprehensive review is given of these studies, particularly concerning infections in skin and in mucosal membranes of the respiratory tract, and of the role of lipids in the antimicrobial activity of breast milk. Based on studies of the microbicidal activities of lipids, both in vitro and in vivo, the possibility of using such lipids as active ingredients in prophylactic and therapeutic dosage forms is considered and examples are given of studies of such pharmaceutical dosage forms in experimental animal models and in clinical trials.     

Dynamic modeling of Listeria monocytogenes growth in pasteurized milk

AIMS: The development and validation of a dynamic model for predicting Listeria monocytogenes growth in pasteurized milk stored at both static and dynamic temperature conditions. METHODS AND RESULTS: Growth of inoculated L. monocytogenes in a commercial pasteurized whole milk product was monitored at various isothermal conditions from 1.5 to 16 degrees C. The kinetic parameters of the pathogen were modelled as a function of temperature using a square root type model, which was further validated using data from 92 published growth curves from eight different milk products. Compared to four published models for L. monocytogenes growth, the model developed in this study performed better, with a per cent discrepancy and bias of 49.1 and -1.01%, respectively. The performance of the model in predicting growth at dynamic temperature conditions was evaluated at four different fluctuating temperature scenarios with periodic temperature changes from -2 to 16 degrees C. The prediction of growth at dynamic storage temperature was based on the square root model in conjunction with the differential equations of the Baranyi and Roberts model, which were numerically integrated with respect to time. The per cent relative errors between the observed and the predicted growth of L. monocytogenes were less than 10% for all temperature scenarios tested. CONCLUSIONS: Available models from experiments conducted in laboratory media may result in significant overestimation of L. monocytogenes growth in pasteurized milk because they do not take into account factors such as milk composition (e.g. natural antimicrobial compounds present in milk) and the interactions of the pathogen with the natural microflora. The product-targeted model developed in the present study showed a high performance in predicting growth of L. monocytogenes in pasteurized milk under both static and dynamic temperature conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature fluctuations often occur during the transportation and storage of pasteurized milk. A high performance, dynamic model for the growth of L. monocytogenes can be a useful tool for effective management and optimization of product safety and can lead to more realistic estimations of pasteurized-milk related safety risks.     

Stability of microbial communities in goat milk during a lactation year: molecular approaches

The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.     

Coexistence of two mitochondrial DNA haplotypes in Japanese populations of Hypera postica (Col., Curculionidae)

Abstract:  In Japan, the alfalfa weevil, Hypera postica , was first recorded in 1982 from Fukuoka and Okinawa Prefectures and has been spreading to many other prefectures. The weevil seriously infests the Chinese milk vetch, Astragalus sinicus , one of the most important honey resources for honeybees in Japan. Direct sequencing of partial mitochondrial DNA and PCR-RFLP data for alfalfa weevil individuals indicated the coexistence of two haplotypes at various localities in Japan. Molecular phylogenetic analysis for H. postica haplotypes and strains indicated that the two Japanese haplotypes had not derived from a single genetic origin. Based on the results, special comments are made on biological control measures using introduced parasitic waSPS.