Immunoglobulins in milk from cows immunized with oral strains of Actinomyces, Prevotella, Porphyromonas, and Fusobacterium.

Immunization of pregnant cows with bacteria leads to the presence of high concentrations of specific antibodies in colostrum and milk. A total of 14 cows was immunized with single strains of heat-killed oral bacteria or pools of strains of Actinomyces, Porphyromonas, Prevotella, and Fusobacterium. Two cows were treated with adjuvant alone. The mean percentages of IgG1, IgG2, IgM, and IgA in all of the milks were 83.8, 3.8, 9.3, and 3.1, respectively. ELISA and whole cell agglutination assays demonstrated high titers in the milks from the cows immunized with either individual strains or the bacterial pools. The highest titers determined by ELISA belonged to the IgG1 isotype and in several milks were 64-fold greater than titers in milk from cows treated with adjuvant alone. The concentrations of all antibodies and the titers determined by ELISA and whole cell agglutination assays markedly decreased from the first to the sixth milkings. The functional specificity of the antibodies was demonstrated by agglutination tests against a wide range of bacteria including members of Actinomyces, Fusobacterium, Porphyromonas, Prevotella, Streptococcus, Eubacterium, Propionibacterium, Peptostreptococcus, Bacteroides, Actinobacillus, Haemophilus, Capnocytophaga, and Wolinella. Minimal cross-reactions with bacteria in other genera were observed with all of the milks. High-titer milk preparations have been obtained from immunized cows, and the capacity of the bovine antibodies to agglutinate target bacteria indicates their potential usefulness in oral passive immunization studies.

Nonspecific antibacterial factors in milk from cows immunized with human oral bacterial pathogens.

Both the immunoglobulins and non-specific antibacterial factors in milk from cows immunized with pathogenic oral bacteria have the potential to influence the oral microflora during passive immunization studies. The first six milks after calving were collected from 2 cows immunized with adjuvant and from 14 cows immunized with adjuvant and heat-killed strains of periodontopathic Actinomyces, Porphyromonas, Prevotella, and Fusobacterium. Analysis of the products from the first to the sixth milks revealed that the protein and lysozyme content decreased approximately 66 and 72%, respectively; the mean specific activity of the enzyme remained relatively constant. In contrast, the mean lactoperoxidase activity increased 2.3-fold in the second milking and increased further in the fourth and sixth milkings. The mean iron-binding activity increased 1.2-fold from the first to the second milkings and then decreased 3.6-fold through the sixth milking. Cows immunized with adjuvant alone showed similar responses. Per unit volume, the milk contained approximately 150 times less lysozyme than whole human saliva obtained from six subjects but higher concentrations of lactoperoxidase and iron-binding components. Purified bovine nonspecific factors prevented the growth of the bacteria used for immunization when bacteria were tested at concentrations similar to those found in saliva and milk. Because bovine nonspecific antibacterial factors could influence both the pathogenic target bacteria and the indigenous microflora in oral passive immunization studies with bovine immunoglobulins, the presence of these proteins should be considered.

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides.

The basis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidase-inactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of muramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe.

Adsorption of lysozyme from human whole saliva by Streptococcus sanguis 903 and other oral microorganisms.

Several strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii plus fresh isolates of Streptococcus salivarius were surveyed for their abilities to deplete lysozyme from human-whole-saliva supernatant. Bacteria were incubated in saliva for 60 min at 37 degrees C and then removed by centrifugation, and the recovered supernatant solutions were assayed for lysozyme activity by using whole cells of Micrococcus lysodeikticus as the substrate. Mean lysozyme depletions by bacterial strains varied over a wide (eightfold) range. The greatest mean depletion of lysozyme (60 to 70%) was observed with S. sanguis (biotype I), serotype b of S. mutans, and the fresh S. salivarius isolates. The lowest mean depletion was noted with S. mitis (15%) and biotype II S. sanguis (ca. 30%). The remaining species and strains exhibited an intermediate degree of depletion. In studies with S. sanguis 903, lysozyme was depleted by normal or heated (90 degrees C, 30 min) bacteria and could be recovered from the organism. Furthermore, under appropriate conditions, lysozyme depletion by cells at 0 and 37 degrees C was very similar. On the basis of these observations, we concluded that depletion was due to the adsorption of lysozyme by the organism. With S. sanguis 903, lysozyme adsorption depended on the concentration of bacteria, time of incubation, and the ionic strength of the medium. The extent of adsorption, however, was independent of pH's of 3.9 to 8.3. When a low concentration of S. sanguis 903 was used, lysozyme adsorption reached saturation (4 mug of adsorbed lysozyme per 10(7) cells) at 20 mug of lysozyme added per ml. Salivary lysozyme adsorption by several other oral microorganisms (A. viscosus WVU 626 and WVU 627, S. sanguis 73x11, S. mutans BHT, and S. salivarius NG) was similar to that of S. sanguis 903 in sensitivity to ionic strength. Lysozyme adsorption by S. sanguis 903 from either a buffer solution or a saliva supernatant was more sensitive to ionic strength at 0 than at 37 degrees C. On the basis of results from experiments in saliva versus buffer, we concluded that saliva had no major effect on the extent of lysozyme adsorption by S. sanguis 903 other than providing a source of ionic strength. A comparison of pH and ionic strength effects on lysozyme adsorption by S. sanguis 903 with literature reports of lysozyme lysis of whole cells and hydrolysis of cell walls, peptidoglycan, and (GlcNAc)(4) suggested that adsorption by S. sanguis 903 was more dependent on electrostatic interactions than was lysozyme catalysis. The possibility is discussed that anionic bacterial surface components mediate lysozyme adsorption and temper the potential effects of lysozyme on the microorganisms.

Doubling time alpha-aminoisobutyrate transport and calcium exchange in cultured fibroblasts from cystic fibrosis and control subjects.

Population doubling time, kinetics of transport of alpha-aminoisobutyrate (AIB) and calcium (Ca) exchange were studied in skin fibroblast monolayers obtained from 5 subjects with cystic fibrosis (CF) and 5 age- and sex-matched controls. Population doubling time as estimated from cell count, protein and DNA was no different in the two groups. KM, Vmax, maximal uptake and time of half maximal uptake of AIB were no different in the two groups. Intracellular Ca pool size based on exchange of 45Ca with unlabelled Ca was significantly greater in monolayers from CF subjects.

Studies of the receptor for phage A25 in group A streptococci: the role of peptidoglycan in reversible adsorption.

Irreversible adsorption of a virulent phage, phage A25, to heat-killed streptococci, groups A, G, and A variant, has been achieved. Adsorption reflected the observed host range for phage A25 in that heat-killed group B cells were not able to inactivate the phage. Broken cells, cell walls, and peptidoglycan prepared from a group A strain K56 failed to adsorb the phage irreversibly, but retained the potential to carry out reversible adsorption. Experimental data including electron microscopy have demonstrated the specificity of reversible adsorption and have identified the peptidoglycan as a necessary cellular component of the receptor. The sensitivity of whole cells and purified peptidoglycan to muralytic enzymes suggests that the cell wall and peptidoglycan must be intact for optimal adsorption. In general the results are explained by postulating that adsorption of A25 phage particles to group A cells occurs by a two-step process; the first step involves recognition and reversible binding of the phage tail to the cell wall peptidoglycan, the second step is an irreversible reaction catalyzed by a yet unidentified cellular component which is destroyed when cells are ruptured.

Reversible inhibition of spore germination by alcohols.

Low levels of alcohols have been found to inhibit the process of spore germination. The extent of germination is dependent upon the concentration of alcohol present in the germinating medium. This inhibition is reversible since removal of the alcohol from the spore environment allows germination to proceed.