Year-long assessment of airborne endotoxin at a concentrated dairy operation

In this study, we monitored total airborne endotoxins at upwind and downwind sites at a large open-lot dairy each month for a year. At the upwind site, the average airborne concentration was 28.5 endotoxin units (EU) m⁻³, while at the downwind edge of the lot and 200 m from the lot edge, the average concentrations were 169 and 72 EU m⁻³, respectively. At the downwind edge of the lot, there was a significant correlation between the airborne endotoxin concentration and wind speed or air temperature. A comparison between total and inhalable airborne endotoxin concentrations, near the end of the study, revealed no significant differences between the two endotoxin collection methods. Our data suggest that endotoxin exposure can be reduced as one increases their distance from the open-lot dairy.     

Detecting mycotoxins in agricultural commodities

It is difficult to obtain precise and accurate estimates of the true mycotoxin concentration of a bulk lot when using a mycotoxin-sampling plan that measures the concentration in a small portion of the bulk lot. A mycotoxin-sampling plan is defined by a mycotoxin test procedure and a defined accept/reject limit. A mycotoxin test procedure is a complicated process and generally consists of several steps: (a) a sample is taken from the lot, (b) the sample is ground (comminuted) in a mill to reduce particle size, (c) a subsample is removed from the comminuted sample, and (d) the mycotoxin is extracted from the comminuted subsample and quantified. Even when using accepted test procedures, there is variability associated with each step of the mycotoxin test procedure. Because of this variability, the true mycotoxin concentration in the lot cannot be determined with 100% certainty by measuring the mycotoxin concentration in a sample taken from the lot. The variability for each step of the mycotoxin test procedure, as measured by the variance statistic, is shown to increase with mycotoxin concentration. Sampling is usually the largest source of variability associated with the mycotoxin test procedure. Sampling variability is large because a small percentage of kernels are contaminated and the level of contamination on a single seed can be very large. Methods to reduce sampling, sample preparation, and analytical variability are discussed.     

百日咳杆菌去内毒素及保护性抗原的纯化

内毒素(Endotoxin,简称ET)是百日咳全菌苗(Bordetellapertussis vaccine)产生副作用的主要毒素之一,且不易除去。现有的分离方法,如蔗糖密度梯度离心法,较繁琐,成本高。本文采用Sepha-cryl S-300凝胶层折法可以简便有效的去除大部分内毒素。初步毒素试验结果表明:已达到日本生物制品规格的要求。两种保护性抗原FHA和LPF-HA也得到进一步分离纯化,为今后研制高效的百日咳组分菌苗提供了实验条件。     

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use.

Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found were Bacillus species and streptococci and 63% of the sera coagulated Limulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain of Escherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Purification and crystallization of insecticidal δ-endotoxin CryIIIB2 from Bacillus thuringiensis

CryIIIB2, an insecticidal protein from Bacillus thuringiensis has been crystallized from 0.6 M NaBr and HEPES buffer at pH 7.0 and X-ray diffraction data collected on a native crystal to 2.4 Å. The insecticidal protein was obtained from a Bacillus thuringiensis (Bt) strain EG7231. Crystals of the endotoxin are orthorhombic, space group C222₁, with unit cell dimensions of a = 122.44, b = and c = Å. A unit cell contains one molecule of the 67,000 Da endotoxin per asymmetric unit. © 1992 Wiley-Liss, Inc.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use

Summary Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found wereBacillus species and streptococci and 63% of the sera coagulatedLimulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain ofEscherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Functional activity of endotoxin binding factors in chronic viral hepatitis B and C

The functional state of cell-mediated and humoral antiendotoxin factors in patients with chronic viral hepatitides B and C has been studied. A decreased content of antibodies to glycolipid of chemotype Re and to Escherichia coli O14 with common enterobacterial antigen was shown to occur in these diseases. In addition, a decreased number of neutrophils, eosinophils and thrombocytes has been noted. The conclusion has been made that patients with chronic hepatitides are not protected from the pathogenic action of endotoxin penetrating from the intestines into the systemic blood stream.     

A simple haemolytic method for quantitation of the delta endotoxin of Bacillus thuringiensis subsp. israelensis from crude samples

A simple haemolytic assay method for quantitative estimation of the delta endotoxin of Bacillus thuringiensis subsp. israelensis from a crude preparation has been developed. The method has several advantages over mosquito-larvicidal methods of assay as it is inexpensive, highly sensitive and easier to run and can be used for performing a reasonably large number of assays rapidly with high precision and with a coefficient of variation that does not exceed 1.96%.     

A simple haemolytic method for quantitation of the delta endotoxin of Bacillus thuringiensis subsp. israelensis from crude samples

A simple haemolytic assay method for quantitative estimation of the delta endotoxin of Bacillus thuringiensis subsp. israelensis from a crude preparation has been developed. The method has several advantages over mosquito-larvicidal methods of assay as it is inexpensive, highly sensitive and easier to run and can be used for performing a reasonably large number of assays rapidly with high precision and with a coefficient of variation that does not exceed 1.96%.     

Augmenting effect of antibiotics on endotoxin activity may cause a safety problem

Enhancing/interfering effect of antibiotics on endotoxin was evaluated using the endotoxin test and the cell line assay in 28SC cells that has a responsiveness consistent with that of human peripheral blood. When a total of 21 products of seven different kinds of antibiotics were tested, none showed any significant effect on the endotoxin test at its therapeutic dose. However, aminoglycosides showed a significant augmenting effect on IL-6 induction of endotoxin in 28SC cells. Detailed examination of the augmenting effect was made on spectinomycin in the in vitro cell line assay and also in the lethal endotoxin challenge assay in D -galactosamine-treated mice. Spectinomycin also enhanced the endotoxin lethality in D -galactosamine-treated mice. A kinetic analysis in endotoxin-sensitized 28SC cells revealed that the augmentation takes place as quickly as 10 min after spectinomycin treatment. Accordingly, a special caution concerning the augmenting effect was assumed necessary for the safety control of antibiotic products as well as for selecting antibiotics for the therapeutic use.     

The fatty acid content of the Bordetella pertussis endotoxin

The fatty acid content of Bordetella pertussis endotoxin has been estimated by several methods. Expressed as 3-hydroxytetradecanoic acid, it was 0.74 mumol (mg lyophilized material)-1, 0.38 mumol being ester-bound, and 0.32 mumol in amide linkage. Reported molar ratios of ester-bound to amide-bound fatty acids in endotoxins of various bacterial species range from 2.4 to 2 in B. pertussis, to 5 to 2 in Salmonella minnesota; according to these figures large differences must exist in the degree of substitution, and the substitution pattern of the glucosaminyl-beta-1,6-glucosamine unit present in the hydrophobic region of endotoxins. When fatty acids, released by acid and alkaline hydrolyses of the B. pertussis endotoxin, were extracted into chloroform, unidentified chromogenic substances appearing in the extract interfered with their colorimetric estimation; no interference was observed when hexane was used instead of chloroform.     

Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features.

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

Aggregates are the biologically active units of endotoxin

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.     

Genome size evolution in relation to leaf strategy and metabolic rates revisited

BACKGROUND AND AIMS: It has been proposed that having too much DNA may carry physiological consequences for plants. The strong correlation between DNA content, cell size and cell division rate could lead to predictable morphological variation in plants, including a negative relationship with leaf mass per unit area (LMA). In addition, the possible increased demand for resources in species with high DNA content may have downstream effects on maximal metabolic efficiency, including decreased metabolic rates. METHODS: Tests were made for genome size-dependent variation in LMA and metabolic rates (mass-based photosynthetic rate and dark respiration rate) using our own measurements and data from a plant functional trait database (Glopnet). These associations were tested using two metrics of genome size: bulk DNA amount (2C DNA) and monoploid genome size (1Cx DNA). The data were analysed using an evolutionary framework that included a regression analysis and independent contrasts using a phylogenetic tree with estimates of molecular diversification times. A contribution index for the LMA data set was also calculated to determine which divergences have the greatest influence on the relationship between genome size and LMA. KEY RESULTS AND CONCLUSIONS: A significant negative association was found between bulk DNA amount and LMA in angiosperms. This was primarily a result of influential divergences that may represent early shifts in growth form. However, divergences in bulk DNA amount were positively associated with divergences in LMA, suggesting that the relationship may be indirect and mediated through other traits directly related to genome size. There was a significant negative association between genome size and metabolic rates that was driven by a basal divergence between angiosperms and gymnosperms; no significant independent contrast results were found. Therefore, it is concluded that genome size-dependent constraints acting on metabolic efficiency may not exist within seed plants.     

Field studies measuring the aerosolization of endotoxin during the land application of Class B biosolids

Endotoxins are a component of Gram-negative bacteria cell walls and are known to be present in biosolids. Endotoxins have been shown to be potent stimulators of the innate immune response causing airway irritation and shortness of breath. Class B biosolids are routinely applied to agricultural lands to enhance soil properties and can be used as an alternative to chemical fertilizers. This study investigated the aerosolized endotoxin dispersed during the land application of Class B biosolids on agricultural land and a concrete surface at two sites in Colorado, USA. Aerosolized endotoxin was captured using HiVol samplers fitted with glass fiber filters, polycarbonate filter cassettes (both open and closed) and BioSampler impinger air samplers. Endotoxins were also measured in the biosolids to allow for correlating bulk biosolids concentrations with aerosol emission rates. Endotoxin concentrations in biosolids, impinger solutions and filter extracts were determined using the kinetic Limulus amebocyte lysate assay. Aerosolized endotoxin concentration was detected from all sites with levels ranging from 0.5 to 642 EU/m³. The four types of sampling apparatus were compared, and the HiVol and open-faced cassette samplers produced higher time-weighted average (TWA) measurements (EU/m³) than the impinger and closed cassette samplers. Ambient wind speed was found to be the variable best describing the observed results with optimal wind speed for highest deposition estimated at 5 m s^?1. It is argued that HiVol air samplers are a particularly reliable approach and subsequent analyses relating TWA measurements to wind speed and biosolids characteristics were based on the measurements collected with those samplers.     

Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene

We measured endotoxin and bacterial levels in tap water, in water purified by reverse osmosis, and in dialysate samples over a 4-month period in a new 10-bed renal dialysis unit. Water treated by reverse osmosis is conducted to the 10 stations through 111 m of piping composed of acrylonitrile butadiene styrene (ABS). All determinations were made prior to the opening of the unit and after the system was purged for 35 h with all bedside station taps open. Formaldehyde disinfection of the piping system was attempted with a recommended protocol after 11 weeks by feeding 2.5 liters of 37% formaldehyde (0.85%, vol/vol) into the delivery system. Prior to water purging, 24 ng of endotoxin per ml was detected. This level decreased to 2.0 ng of endotoxin after the purging. Levels of endotoxin remained below 1.0 ng of endotoxin per ml throughout the duration of the study. In contrast, the level of viable microorganisms recovered from the treated water was approximately 3.5 X 10(4) CFU/100 ml. Even after disinfection of the system, there was no significant decrease in culturable bacteria from the water even though endotoxin levels were lower. Species isolated from the renal dialysis system were predominately pseudomonads, whereas species isolated from the tap water were Bacillus and Flavobacterium species. ABS provides a surface suitable for long-term colonization and growth of bacteria. Currently recommended decontamination protocols are ineffective in removing potentially pathogenic bacteria from ABS pipes and thus constitute an increased risk to patients undergoing dialysis.     

Characterization of Virus- and Endotoxin-induced Interferons Obtained from the Serum and Urine of Rabbits

Interferons induced in the rabbit by Newcastle disease virus or by endotoxin have been further characterized as to their physicochemical stability and molecular size by Sephadex G-100 gel filtration. Endotoxin-induced interferon obtained from serum was more labile than virus-induced interferon. Both endotoxin and virus induced interferons obtained from serum contained two peaks: a minor high molecular weight (>100,000) peak and a major lower molecular weight peak. The molecular weight of the major peak induced by endotoxin was 54,000, and that induced by Newcastle disease virus was 46,000. The gel filtration pattern of interferon recovered from the urine of animals inoculated with virus reflected faithfully the pattern found in serum except that there was proportionately less of the high molecular weight peak. However, the urine interferon from endotoxin-inoculated animals contained only one broad peak with a molecular weight of 35,000. This was not the peak fraction present in the serum of such animals. It is postulated that this may represent the basic unit of endotoxin-induced interferon, and that the serum components are either polymers or conjugates of this basic unit.     

Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene.

We measured endotoxin and bacterial levels in tap water, in water purified by reverse osmosis, and in dialysate samples over a 4-month period in a new 10-bed renal dialysis unit. Water treated by reverse osmosis is conducted to the 10 stations through 111 m of piping composed of acrylonitrile butadiene styrene (ABS). All determinations were made prior to the opening of the unit and after the system was purged for 35 h with all bedside station taps open. Formaldehyde disinfection of the piping system was attempted with a recommended protocol after 11 weeks by feeding 2.5 liters of 37% formaldehyde (0.85%, vol/vol) into the delivery system. Prior to water purging, 24 ng of endotoxin per ml was detected. This level decreased to 2.0 ng of endotoxin after the purging. Levels of endotoxin remained below 1.0 ng of endotoxin per ml throughout the duration of the study. In contrast, the level of viable microorganisms recovered from the treated water was approximately 3.5 X 10(4) CFU/100 ml. Even after disinfection of the system, there was no significant decrease in culturable bacteria from the water even though endotoxin levels were lower. Species isolated from the renal dialysis system were predominately pseudomonads, whereas species isolated from the tap water were Bacillus and Flavobacterium species. ABS provides a surface suitable for long-term colonization and growth of bacteria. Currently recommended decontamination protocols are ineffective in removing potentially pathogenic bacteria from ABS pipes and thus constitute an increased risk to patients undergoing dialysis.