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The following is a review of some of the work that we have done since 2007 regarding the importance of molds in the phenomenon of sick building syndrome (SBS). In these studies we first examined mold contamination in air handling units (AHU). Our results showed that Cladosporium sp. were commonly recovered in AHU as growth sites and free spores. They were found mainly on the blower wheel fan blades, the ductwork, and cooling coil fans. Our results showed that the presence of species of molds other than Cladosporium in locations other than the blower wheel blades indicated that the AHU condition was not optimal. In a series of three papers, we examined growth and mycotoxin production by Chaetomium globosum (CG). In these studies we showed that CG produces two potent mycotoxins, chaetoglobosin A (Ch-A) and chaetoglobosin C (Ch-C) when grown on building material. We discovered that these toxins break down when exposed to temperatures in excess of 75 degrees C. We also showed that growth and mycotoxin production by CG is favored at a neutral pH. In another study, we showed that mycotoxins can be detected in body fluids and human tissues from patients exposed to mycotoxin producing molds, and we showed which human tissues or fluids were the most likely to give positive results for detection of these compounds. Finally, we showed that the macrocyclic trichothecene mycotoxins (MTM) produced by Stachybotrys chartarum (SC) are detectable in experimental animals soon after exposure and we described the dynamics of MTM tissue loading.

Macrocyclic trichothecenes, mycotoxins produced by Stachybotrys chartarum, have been implicated in adverse reactions in individuals exposed to mold-contaminated environments. Cellular and humoral immune responses and the presence of trichothecenes were evaluated in patients with mold-related health complaints. Patients underwent history, physical examination, skin prick/puncture tests with mold extracts, immunological evaluations and their sera were analyzed for trichothecenes. T-cell proliferation, macrocyclic trichothecenes, and mold specific IgG and IgA levels were not significantly different than controls; however 70% of the patients had positive skin tests to molds. Thus, IgE mediated or other non-immune mechanisms could be the cause of their symptoms.

The following is a review of some of the work we have done since 2004 regarding the importance of molds and their mycotoxins in the phenomenon of sick building syndrome (SBS). In these studies we showed that the macrocyclic trichothecene mycotoxins (MTM) of Stachybotrys chartarum (SC) are easily dissociated from the surface of the organism as it grows and could therefore be consequently spread in buildings as the fungus experiences additional water events. We then showed that SC and Penicillium chrysogenum (PC) colonies remain viable long after a water source has been removed, and the MTM produced by SC remain toxic over extended periods of time. We next showed that PC when inhaled, can release in vivo, a protease allergen that can cause a significant allergic inflammatory reaction in the lungs of mice. We then showed, in a laboratory study, that the MTM of SC can become airborne attached to spores or SC particulates smaller than spores. Following that study, we next showed that the same phenomenon actually occurred in SC infested buildings where people were complaining of health problems potentially associated with SBS. Finally, we were able to demonstrate the presence of MTM in the sera of individuals who had been exposed to SC in indoor environments. This last study was done with enough mold exposed individuals to allow for the statistical significance of SC exposure to be evaluated.

This study demonstrates the detection and dynamics of macrocyclic trichothecene mycotoxin (MTM) tissue loading using a commercially available assay in a goat model. The detection of MTMs has been difficult and complex due to the uncertainty of what tissues to examine and when to sample. Twelve goats (two groups of each) were instilled with Stachybotrys chartarum conidial suspension via the trachea. The first group was challenged repeatedly with fungal conidia containing 1 mg/kg of MTM per instillation whereas the second group was exposed once, to spores with a calculated concentration of 5 µg/kg of mycotoxin. These toxin estimates were generated by the QuantiToxTM Kit assay; a conidium of S. chartarum possessed 8.5 pg of MTM. After repeated exposure of 3 days, MTM was detected in one of six animals. This animal and two others from the same group had mycotoxin detected in their serum 24 hours after challenge at a comparable level (1.69 ng/mL) to the six animals challenged with a single dose (2.02 ng/mL) at the same time post-instillation. Results showed that MTMs are detectable in experimental animals soon after challenge and contribute to the understanding of the role of these mycotoxins in the disease process following mold exposure.

The growth of indoor molds and their resulting products (e.g., spores and mycotoxins) can present health hazards for human beings. The efficacy of chlorine dioxide gas as a fumigation treatment for inactivating sick building syndrome-related fungi and their mycotoxins was evaluated. Filter papers (15 per organism) featuring growth of Stachybotrys chartarum , Chaetomium globosum , Penicillium chrysogenum , and Cladosporium cladosporioides were placed in gas chambers containing chlorine dioxide gas at either 500 or 1,000 ppm for 24 h. C. globosum was exposed to the gas both as colonies and as ascospores without asci and perithecia. After treatment, all organisms were tested for colony growth using an agar plating technique. Colonies of S. chartarum were also tested for toxicity using a yeast toxicity assay with a high specificity for trichothecene mycotoxins. Results showed that chlorine dioxide gas at both concentrations completely inactivated all organisms except for C. globosum colonies which were inactivated an average of 89%. More than 99% of ascospores of C. globosum were nonculturable. For all ascospore counts, mean test readings were lower than the controls ( P < 0.001), indicating that some ascospores may also have been destroyed. Colonies of S. chartarum were still toxic after treatment. These data show that chlorine dioxide gas can be effective to a degree as a fumigant for the inactivation of certain fungal colonies, that the perithecia of C. globosum can play a slightly protective role for the ascospores and that S. chartarum , while affected by the fumigation treatment, still remains toxic.

The efficacy of chlorine dioxide (ClO2) in detoxifying two potential bioterrorism agents, the trichothecene mycotoxins verrucarin A and roridin A, was evaluated. In the first experiment, verrucarin A (1, 5, or 10 μg) and roridin A (5 or 10 μg) were each inoculated onto square-inch sections of glass, paper, and cloth and exposed to 1000 ppm of ClO2 for either 24 or 72 h at room temperature. In the second experiment, verrucarin A and roridin A (1 or 2 ppm in water) were treated with 200, 500, or 1000 ppm ClO2 for up to 116 h at room temperature in light and dark conditions ( N = 9 per treatment for test and control). A yeast assay using Kluyveromyces marxianus was used to quantify the toxicity of verrucarin A and roridin A. Additionally, high-performance liquid chromatography was performed on selected samples. Results for the first experiment showed that ClO2 treatment had no detectable effect on either toxin. For the second experiment, both toxins were completely inactivated at all tested concentrations in as little as 2 h after treatment with 1000 ppm ClO2. For verrucarin A, an effect was seen at the 500 ppm level, but this effect was not as strong as that observed at the 1000 ppm level. Roridin A toxicity was decreased after treatment with 200 and 500 ppm ClO2, but this was not significant until the 24-h exposure time was reached. These data show that ClO2 (in solution) can be effective for detoxification of roridin A or verrucarin A at selected concentrations and exposure times.

Highly respirable particles (diameter

Forty-two, mixed-sex, weanling goats were randomly allotted to six treatment groups: principal autoclaved dust ( n = 6), control non-autoclaved dust (n = 6), principal dry-heat dust (n = 6), and control non-dry-heat dust (n = 6). Principals were treated with appropriate dust for one 4 h treatment in a closed tent. The data from the principal dust group ( n = 9) and the control non-dust group (n = 9) were recorded after one 4 h dust treatment prior to the start of the present study. The endotoxin (ET) concentrations were determined for autoclaved dust (13.3g ET/g), dry-heated dust (0.173g ET/g), and non-treated dust 26.9g ET/g. The tent aerosolized dust concentrations were: autoclaved dust 0.369 g/(m 3 min) with 4.904g ET/(m 3 min); dry-heated dust 0.347 g/(m 3 min) with 0.0015g ET/(m 3 min); and non-treated dust 0.539 g/(m 3 min) with 4.904g ET/(m 3 min). These ET aerosol concentrations caused the autoclave dust goat group and the non-treated dust goat group to significantly increase their rectal temperatures at 4, and 8 h and total white blood cells (WBCs) increased at 12 and 24 h compared to their respective non-dust control groups. The dry-heat aerosol dust ET concentration in the tent did not induce an increased mean rectal temperature response or an increased mean total WBC count. Of the three principal dust products only the non-treated dust contained viable microbes. Published by Elsevier Science B.V.

Weanling female Spanish goats ( n =36) were randomly allotted to four treatment groups (each group, n =9): principal dust group with antibiotic; principal dust group without antibiotic; control group with antibiotic; and control group without antibiotic. The principals were exposed to twenty-two 4 h dust treatments in a closed tent. Dust treatments occurred as follows: one dust treatment, 18-day rest, seven daily dust treatments, 7-day rest, seven daily dust treatments, 7-day rest, and seven daily dust treatments. All principals and six controls were euthanatized and necropsied at the end of the experiment. There was an interaction between dust and tilmicosin antibiotic for feed intake and average daily gain following the first dust treatment. The interaction occurred because control goats not exposed to dust nor receiving tilmicosin protection had lower ( P P P ≤0.0003). Four hours ( P ≤0.0016) after dust treatment, the mean rectal temperature of the two principal groups was higher than the controls. On the first 7-day dust treatment series there was a significant ( P ≤0.0006) difference between the groups. The mean rectal temperatures of the two principal groups at 8 h on day 1 were significantly ( P ≤0.02) higher than the control groups. On the second 7-day dust treatments, there were no significant differences in mean rectal temperature between the groups. On the third 7-day treatment series there were significant ( P ≤0.0001) differences among the four groups. On the first day of the dust treatment series, at 8 h, the trend of the principal groups’ mean rectal temperature was higher than the controls. It appears that repeated endotoxin/dust exposures induces a state of tolerance for increased rectal temperatures that are characteristic after one dust exposure. There were no gross observable differences between the controls and principal respiratory tracts. The following histologic changes were observed in the principal goats: a mild alveolar septal hypercellularity, moderate multifocal airway exudation of neutrophils and macrophages containing foreign particulate material, and there was a marked increase in bronchial associated lymphoid tissue compared to controls. The diagnosis was mild acute exudative bronchointerstital pneumonia. No significant microscopic lesions were observed in the control goats.

Ninety-one isolates of Pasteurella multocida (Pm) and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34 (27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories.

Over the past 20 years, exposure to mycotoxin producing mold has been recognized as a significant health risk. Scientific literature has demonstrated mycotoxins as possible causes of human disease in water-damaged buildings (WDB). This study was conducted to determine if selected mycotoxins could be identified in human urine from patients suffering from chronic fatigue syndrome (CFS). Patients (n = 112) with a prior diagnosis of CFS were evaluated for mold exposure and the presence of mycotoxins in their urine. Urine was tested for aflatoxins (AT), ochratoxin A (OTA) and macrocyclic trichothecenes (MT) using Enzyme Linked Immunosorbent Assays (ELISA). Urine specimens from 104 of 112 patients (93%) were positive for at least one mycotoxin (one in the equivocal range). Almost 30% of the cases had more than one mycotoxin present. OTA was the most prevalent mycotoxin detected (83%) with MT as the next most common (44%). Exposure histories indicated current and/or past exposure to WDB in over 90% of cases. Environmental testing was performed in the WDB from a subset of these patients. This testing revealed the presence of potentially mycotoxin producing mold species and mycotoxins in the environment of the WDB. Prior testing in a healthy control population with no history of exposure to a WDB or moldy environment (n = 55) by the same laboratory, utilizing the same methods, revealed no positive cases at the limits of detection.

The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme.

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 10(4) copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.

The properties of an extracellular neuroaminidase produced by a Pasteurella haemolytica A1 strain (isolated from a case of bovine pneumonia) during growth in a defined medium were examined in this investigation. This enzyme, isolated from concentrated culture supernatants of P. haemolytica A1, was active against N-acetylneuramin lactose, human alpha 1-acid glycoprotein, fetuin, and bovine submaxillary mucin. Neuraminidase production paralleled bacterial growth in a defined medium and was maximal in the stationary phase of growth. The enzyme was purified to homogeneity by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 100.62 mumol of sialic acid released per min per mg of protein against human alpha 1-acid glycoprotein. The Km value for this enzyme with human alpha 1-acid glycoprotein as the substrate was 1.1 mg/ml, and the enzyme possessed a pH optimum of 6.5. The P. haemolytica A1 neuraminidase had a molecular weight of approximately 150,000 as estimated by gel filtration and approximately 170,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at 4 degrees C for 3 h. At 37 degrees C for 3 h, 25% of enzymatic activity was lost. Approximately 55% of the enzyme activity was lost within 30 min at 50 degrees C, with greater than 70% of the enzyme activity being destroyed within 10 min at temperatures of > or = 65 degrees C.

Drywall from China has been reported to release sulfur producing products which are corrosive to metals, result in noxious odors, and represent a significant health risk. It has been reported that these emissions produce medical symptoms such as respiratory or asthma type problems, sinusitis, gastrointestinal disorders, and vision problems in home owners and their household pets. We report here a method of identifying a causative agent for these emissions by sampling affected gypsum wallboard and subjecting those samples to Real Time Polymerase Chain Reaction [RT-PCR] studies. Specific DNA probes and primers have been designed and patented that detect a specific iron and sulfur reducing bacterium (i.e., Thiobacillus ferrooxidans). One hundred percent of affected drywall samples obtained from homes located in the southeastern United States tested positive for the presence of T. ferrooxidans. All negative controls consisting of unaffected wallboard and internal controls, Geotrichum sp., tested negative within our limits of detection.

The goal of this study was to determine if selected mycotoxins (trichothecenes, aflatoxins, and ochratoxins) could be extracted and identified in human tissue and body fluids from patients exposed to toxin producing molds in their environment. Human urine and methanol extracted tissues and sputum were examined. Trichothecenes were tested using competitive ELISA techniques. Aflatoxins B1, B2, G1, and G2, and ochratoxin A were tested by using immunoaffinity columns and fluorometry. Test sensitivity and specificity were determined. Levels of detection for the various mycotoxins varied from 0.2 ppb for trichothecenes, 1.0 ppb for aflatoxins, and 2.0 ppb for ochratoxins. Trichothecene levels varied in urine, sputum, and tissue biopsies (lung, liver, brain) from undetectable ( 10.0 ppb. Negative control patients had no detectable mycotoxins in their tissues or fluids. These data show that mycotoxins can be detected in body fluids and human tissue from patients exposed to mycotoxin producing molds in the environment, and demonstrate which human tissues or fluids are the most likely to yield positive results.

Chaetomium globosum is frequently isolated in water-damaged buildings and produces two mycotoxins called chaetoglobosins A and C when cultured on building material. In this study, the influence of ambient pH on the growth of C. globosum was examined on an artificial medium. This fungus was capable of growth on potato dextrose agar ranging in pH from 4.3 to 9.4 with optimal growth and chaetoglobosin C production occurring at a neutral pH. In addition, our results show that sporulation is favored in an acidic environment.

Chaetomium globosum is commonly found in water-damaged buildings and produces the mycotoxins chaetoglobosin A and chaetoglobosin C (Ch-A and Ch-C, respectively). While attempting to purify Ch-A and Ch-C, we observed that these mycotoxins were broken down after heating. The objective of this study was to determine the temperature and the amount of time necessary to break down Ch-A and Ch-C. We demonstrated that the amounts of Ch-A were significantly reduced when exposed to 75 degrees C for 24 h and 100 degrees C for 90, 120, or 150 min. Under the same conditions, the levels of Ch-C were also lower (although not significantly). At 175 degrees C, no Ch-A was detected after 15 min and Ch-C was significantly reduced after 30 min. Our findings will aid other researchers who work with these mycotoxins in the future.

Six clinical isolates of Pseudomonas cepacia (representing the five serotypes of the organism) were examined for the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO) in their lipopolysaccharide (LPS). Purified LPS was examined for the presence of KDO by the thiobarbituric acid (TBA) assay and by gas chromatography. All strains possessed KDO. One strain possessed KDO that was detectable by the TBA assay after mild acid hydrolysis with 0.04 M H2SO4 at 100 degrees C for 20 min. The other strains also possessed KDO but it was only demonstrable by the TBA assay after strong acid hydrolysis (4 M HCl for 60 min at 100 degrees C). All six purified LPS preparations were shown to possess KDO by two separate gas chromatography procedures. LPS isolated from the six strains of P. cepacia was toxic for mice.

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.

To date, no study has effectively demonstrated a direct human exposure to mycotoxins in mold-contaminated buildings. Therefore, the authors investigated the presence of trichothecene mycotoxins in sera from individuals exposed to indoor molds (specifically Stachybotrys chartarum). Sera from occupants of contaminated (test samples, n=44) and uncontaminated (control samples, n=26) buildings were analyzed using a competitive enzyme-linked immunosorbent assay (ELISA) highly specific for macrocyclic trichothecenes. Twenty-three samples were significantly different (p < 0.05) from normal human serum tested in the same manner, whereas only 1 of the control samples tested positive. Mass spectrometry analysis could not confirm the presence of intact S. chartarum macrocyclic trichothecenes. The authors hypothesize that this result was caused by uncharacterized ELISA-reactive metabolic breakdown products. Data from this study suggest that trichothecene mycotoxins can be demonstrated in the tissues of certain individuals exposed to S. chartarum in contaminated buildings.

Objective—To determine effects of repeated aerosol exposures to fly ash dust on respiratory tracts of tent-confined goats. Animals—12 weanling Boer-Spanish crossbred goats. Procedure—Goats were randomly assigned to 2 groups: fly ash treatment group (principal goats, n = 6) or control group (control goats, 6). Aerosolized fly ash dust was provided during a 4-hour period for each of 6 applications given over 3 months and one 2-hour application prior to necropsy. Fly ash particle diameters ranged from 0.1 to 130 µm and averaged 17.8 µm, with 1.5% of fly ash particles in the 0.1- to 5-µm-diameter range. A mean ± SD of 748 ± 152 g/treatment was delivered inside a tent containing principal goats; control goats were placed inside a similar tent for 4-hour treatments without dust. Following treatment, rectal temperatures were taken at 0, 4, 6, 8, 24, and 72 hours; Hcts were recorded at 0, 24, and 72 hours. Results—Rectal temperatures were significantly increased at 4, 6, and 8 hours and decreased at 72 hours, compared with 0 hours. Mean ± SEM Hct values were significantly increased for principal goats (37.47 ± 0.39%), compared with control goats (36.17 ± 0.42%). A significant increase in the mean area of gross atelectatic lung lesions (1,410 mm2) was found in principal goats (n = 6), compared with control goats (440 mm2; 5). Conclusions and Clinical Relevance—An increase in atelectatic lung lesions was observed in principal goats, compared with control goats; however, overall, fly ash dust effects were nontoxic. ( Am J Vet Res 2005;66:991–995)

Recent studies have correlated the presence of Stachybotrys chartarum in structures with SBS. S. chartarum produces mycotoxins that are thought to produce some of the symptoms reported in sick-building syndrome (SBS). The conidia (spores) produced by Stachybotrys species are not commonly found in the air of buildings that have been found to contain significant interior growth of this organism. This could be due in part to the large size of the Stachybotrys spores, or the organism growing in hidden areas such as wall cavities. However, individuals in buildings with significant Stachybotrys growth frequently display symptoms that may be attributed to exposure to the organism's mycotoxins. In addition, Stachybotrys colonies produce a "slime" or polysaccharide (carbohydrate) matrix that coats the hyphae and the spores. The intent of this project was to determine whether the carbohydrate matrix and the mycotoxins embedded in it could be removed from the spores by repeated washings with either aqueous or organic solvents. The results demonstrated that the process of spore washing removed compounds that were toxic in a protein translation assay as compared to spores that were washed with an organic solution, however a correlation between carbohydrate removal during the washing process and the removal of mycotoxins from the spore surface was not observed. These data demonstrated that mycotoxins are not likely to be found exclusively in the carbohydrate matrix of the spores. Therefore, mycotoxin removal from the spore surface can occur without significant loss of polysaccharide. We also showed that toxic substances may be removed from the spore surface with an aqueous solution. These results suggest that satratoxins are soluble in aqueous solutions without being bound to water-soluble moieties, such as the carbohydrate slime matrix.

The adult mouse model was employed to examine the mechanism of virulence enhancement by the extracellular type-specific antigen (ETSA) of type-IIIStreptococcus agalactiae. Intraperitoneal injection of ETSA along with the infecting strain lowered the LD50 value forS. agalactiae and allowed for a more rapid proliferation of the organisms as opposed to that caused by the presence of a nonspecific dextran. The ETSA did not hinder recruitment of peritoneal exudate cells (PEC) into the peritoneal cavity as has been shown with capsular antigens of other bacteria. Chemiluminescent responses of mouse resident PEC demonstrated that these cells could render a respiratory burst when exposed to type-IIIS. agalactiae in the absence of complement and/or type-specific antibody. If the mouse PEC were exposed to ETSA before phagocytosis was attempted, however, ingestion of the type-III group-B streptococci was reduced. ETSA did not affect the viability of the PEC as determined by trypan blue exclusion. These results indicate that the ETSA can affect mouse PEC in an unidentified manner, thus rendering them less capable of ingesting and/or kiling type-IIIS. agalactiae.

Six strains of viridans streptococci isolated from confirmed cases of subacute bacterial endocarditis were studied for production of extracellular material. All six strains, when grown to the exponential phase, produced exoproducts that had similar elution profiles on a G-100 Sephadex column. Since essential nutrients, such as amino acids, may be periodically growth limiting to streptococci in the fibrin-covered lesions on heart valves, the potential to elaborate extracellular protein and other material by streptococci that were deprived of essential amino acids was studied. Examination of supernatant fluids from cultures of Streptococcus MG intermedius deprived of glutamate and cystine revealed the presence of a complex mixture of extracellular materials in amounts comparable to those produced by normallly growing cells, Although only a slight (21 to 24%) increase in total protein occurred during amino acid deprivation of 12 h, the extracellular material contained numerous protein components, several of which demonstrated proteolytic activity. On a cell dry weight basis, the amino acid-deprived cells produced four-to eightfold more protease(s) than did exponential cells grown in complete medium. These results demonstrate that viridans streptococci are capable of elaborating potentially damaging compounds even when their multiplication has been arrested by nutritional deprivation.

The properties of an extracellular neuraminidase produced by a Streptococcus sanguis strain (isolated from a confirmed case of subacute bacterial endocarditis) during growth in a defined medium was examined in this investigation. This enzyme, isolated from concentrated culture supernatants of S. sanguis biotype II, was active against human alpha-1 acid glycoprotein, N-acetylneuramin lactose, bovine submaxillary mucin, and fetuin. Neuraminidase production paralleled bacterial growth in defined medium and was maximal in the early stationary phase of growth but decreased dramatically, probably owing to protease production, during the late stationary phase. The enzyme was purified to near homogeneity by a combination of salt fractionation, ion-exchanged chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 174.4 mumol of sialic acid released per min per mg of protein against human alpha-1 acid glycoprotein. The Km value for this enzyme with human alpha-1 acid glycoprotein as substrate was 2.5 X 10(-3) M, and the enzyme possessed a pH optimum of 6.5. The S. sanguis neuraminidase had a molecular weight of approximately 85,000 as estimated by gel filtration and approximately 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at temperatures of 4 and 37 degrees C for 3 h, but approximately 50% of the enzymatic activity was lost within 30 min at 50 degrees C, with 100% of the enzymatic activity being destroyed within 10 min at temperatures of greater than or equal to 65 degrees C.

Extracellular neuraminidase from a type III fresh clinical isolate of a group B streptococcus was purified by a combination of salt fractionation, affinity chromatography of Affi-Gel blue, ion-exchange chromatography on diethylaminoethylcellulose, and gel filtration on Sephacryl S-200. These procedures yielded enzyme which was purified approximately 1,000-fold compared with the enzyme found in the original supernatant fluid. This type III streptococcal neuraminidase had a molecular weight of approximately 125,000 as estimated by filtration on Sephacryl S-200 and approximately 106,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to the majority of other bacterial neuraminidases, the type III group B streptococcal enzyme had no effect on colominic acid or N-acetylneuramin-lactose; however, it was quite active on bovine submaxillary mucin.

Recent evidence suggests that colonization of the upper respiratory tract by gram-negative bacilli is mediated by adherence to regional epithelial cells. Buccal epithelial cells were obtained for study from 12 seriously ill patients, all of whom were colonized with Pseudomonas aeruginosa. In comparison to cells from uncolonized controls, cells obtained from these patients attached significantly more P. aeruginosa organisms during incubation in vitro. Although the sialic acid content of colonized patients' cells was less than that of controls' cells, removal of sialic acid from normal cells with neuraminidase did not increase bacillary adherence. Trypsinization of normal cells increased bacillary adherence and significantly reduced the amount of fibronectin on the cell surface. Both trypsinized normal cells and cells recovered from seriously ill colonized patients attached large numbers of P. aeruginosa organisms in vitro and demonstrated decreased fibronectin on the cell surface by immunofluorescent staining. These findings suggest that the host alteration associated with increased susceptibility to adherence by P. aeruginosa is the loss of fibronectin from the cell surface.

The purpose of these studies was to determine if testicular macrophages are capable of bactericidal activity. Testicular macro phages were isolated from adult Wistar rats and studied in vitro. Studies were designed to determine if these cells could kill pathogenic gram-negative organisms and if these cells secreted lysozyme, an enzyme involved with the lysis of the cell wall of gram-positive bacteria. The regulation of lysozyme secretion by hormones and lipopolysaccharide was also studied. The secretion of this enzyme by testicular macrophages was also compared to enzyme secretion by macrophages isolated from other tissues. We also studied the secretion of superoxide anion, which is known to be involved in cytotoxic reactions. It was found that testicular macrophages were capable of killing up to approximately 38% of a virulent encapsulated strain of Kiebsiella pneumoniae within 1 h. This process was in part dependent upon the presence of immune serum generated against these organisms but could not be mimicked by control serum or immune serum tested in the absence of macrophages. Testicular macrophages secreted lysozyme in culture for at least 8 days; however, macro phages from the peritoneal cavity and lung secreted significantly more lysozyme under the same conditions. Lipopolysaccharide suppressed lysozyme secretion in a dose-dependent manner, whereas neither follicle-stimulating hormone, testosterone, nor leuteininzing hormone had an effect on lysozyme secretion. Finally, testicular macrophages secreted superoxide anion in a manner similar to peritoneal macro phages. These studies indicate that testicular macrophages have the capability to mount an appropriate defense against pathogenic bacteria by opsonizationdependent phagocytosis, the secretion of lysozyme, and the production of super oxide anion.

A simple and rapid procedure for the isolation in high yield (about a 30% recovery based on the total 30 to 60% ammonium sulfate recovery) of homogeneous purified group A streptococcal M-protein is described. M-proteins extracted from whole cells of group A streptococci by treatment with hot HCl were neutralized, fractionated with ammonium sulfate, dialyzed, lyophilized, and then subjected to treatment with hot 60% trichloroacetic acid. This was shown to produce an M-protein preparation, free of group A carbohydrate activity and extraneous antigens, in yields up to 10-fold higher than previous methods in about one-fifth the time. These M-protein preparations were shown to: (i) have similar amino acid compositions to their respective type-specific proteins purified by diethylaminoethyl and O -(carboxymethyl) cellulose chromatography, (ii) react with their respective type-specific antisera in Ouchterlony diffusion, (iii) produce antisera in rabbits capable of promoting streptococcal long-chain formation in vitro, and (iv) give only one major band on polyacrylamide gel disk electrophoresis. The data allow for an explanation of the hitherto described multiple banding M-proteins seen on acrylamide electrophoresis.

Two sialic acid-containing type III group B streptococcal antigens were obtained from a supernatant growth medium, purified by anion exchange or gel filtration, and found to be free of group B reactivity. Quantitation of the high-molecular-weight extracellular type III antigen indicated that approximately 20-fold more antigen was recoverable from the growth medium than could be obtained by neutral buffer extraction of whole cells.

Short term, carrier mediated transport of D-glucose, L-leucine and L-lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two-limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a Km of 0.1 mM for L-leucine and 0.05 mM for L-lysine; Vmax values were 2.0 and 2.85 nmole/mg protein/90 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat-killed staphylococci by phagocytosis stimulated 2-deoxy-D-glucose (2-dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2-dOG increased in post-phagocytic macrophages, the kinetics of the system were not altered significantly. The Km for both pre- and post-phagocytic transport of 2-dOG was shown to be 1.2 mM and the Vmax was shown to increase from a pre-phagocytic value of 20 nmoles/mg protein/90 seconds to a post-phagocytic 27 nmoles/mg protein/90 seconds. Phagocytosis of heat-killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages.

The release of serotype III group B streptococcal polysaccharides into the supernatant fluid was examined under a variety of physiological conditions. Release of both high- and low-molecular-weight type III antigens was fairly constant throughout exponential growth, but increased markedly upon entering the stationary phase of growth. Increased glucose and decreased phosphate concentrations both caused a large increase in release of antigens. Inhibition of protein synthesis in exponentially growing cells by chloramphenicol (10 micrograms/ml) caused a condition of unbalanced growth in which antigen release was increased greatly over control values. Strain variability in antigen release was also observed. Strains which are known to be high neuraminidase producers released elevated levels of both low- and high-molecular-weight type III antigens. Non-neuraminidase-producing strains released considerably less high-molecular-weight antigen, but similar levels of the low-molecular-weight antigen compared with the high neuraminidase producers. Strain D136C, a type III non-neuraminidase producer, released negligible quantities of the high-molecular-weight antigen in the supernatant fluid. These results indicate that both the physiological environment and the type III strain are important in determining the quantity of type-specific antigen released into the culture fluid.

The level of total extracellular neuraminidase produced by 74 clinical isolates of group B streptococci isolated from diseased or asymptomatically colonized infants was assayed. Extracellular neuraminidase was obtained from concentrated filtrates of exponentially growing cultures of group B streptococci grown in a chemically defined medium (FMC) containing supplemental protein. The total activity of extracellular enzyme produced by these clinical isolates ranged from less than 10 to 360 nmol of sialic acid released per min per mg of cell dry weight. Strains were arbitrarily classified as either nonproducers (less than 10 nmol/min per mg of cell dry weight), low producers (greater than 10 to less than or equal 140 nmol/min per mg of cell dry weight), or high producers (greater than 140 to 360 nmol/min per mg of cell dry weight). Type III isolates from diseased infants were significantly more often classified as high producers than strains of group B streptococci of other serotypes from diseased infants (P less than 0.001). Furthermore, the serotype III strains isolated from neonatal infections were more often high producers than those of the same serotype from asymptomatically colonized infants (P less than 0.025). These results suggest that the ability to produce elevated levels of neuraminidase may be related to the frequent association of type III strains with disease among neonates.

As the variant surface coat glycoprotein (VSG) was shed from Trypanosoma brucei rhodesiense into the blood of infected rats, it was biochemically characterized and compared with VSG that had been purified from trypanosomal homogenates. To determine if VSG was in association with lipid, membranes and lipoproteins in plasma of infected rats (IRP), VSG isolated from plasma (PVSG), and VSG isolated from trypanosomal homogenates (HVSG) were all concentrated by ultracentrifugation and assayed for the presence of VSG by radial immunodiffusion (minimum level of detection, 25 micrograms/ml) and by immunoelectroblots (minimum level of detection, 1 microgram/ml). Crimson red was used to detect lipid (minimum level of detection, 10 micrograms per sample) in electrophoresed samples. The VSG was neither concentrated with membrane or lipoprotein fractions nor stained by lipid crimson. Lipids from normal rat plasma, IRP, trypanosomal homogenates, HVSG, and PVSG were also extracted and separated by thin-layer chromatography (minimum level of detection, 20 micrograms of trypanosomal phospholipid per sample). The trypanosomal homogenates had five bands as detected by iodine vapors, of which three were phospholipids as detected by molybdenum blue. Both normal rat plasma and IRP had identical patterns of bands with a single phospholipid. The PVSG had one neutral lipid contaminant that apparently was not physically associated with the shed surface coat. The HVSG contained no lipids at all. Therefore, no evidence was obtained to implicate an association between membranes and VSG, once the latter had been shed into the blood of infected hosts. From immunoelectroblots of denatured material, it was determined that both HVSG and PVSG had the same reduced molecular weight. From molecular sieve column chromatography, however, it was determined that VSG released during the homogenization of trypanosomes is a noncovalently linked dimer, whereas that shed in the blood is apparently a trimer. This difference in native structure made no difference in immunological effect. Administered in a regimen that mimicked what the host encounters during a first peak of parasitemia, both HVSG and PVSG induced nonspecific proliferation of splenic lymphocytes and production of unelicited antibodies without the generation of nonspecific immunosuppression. This polyclonal activation of lymphocytes was not the result of contamination by exogenous pyrogen, because the activity was lost if VSG was immunologically absorbed from plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Cryptococcus neoformans cell and culture supernatant extracts were fractionated by ion exchange and gel filtration column chromatography. Various fractions were used to immunize mice, and to assess the release of migration inhibition factor, delayed type hypersensitivity, and protective immunity after challenge with C. neoformans. Results suggest that the C. neoformans fractions, which protect mice, contain a high molecular weight, predominantly carbohydrate antigen that can be distinguished from the capsular polysaccharide.

Ten different group A streptococcal M-protein preparations purified by trichloroacetic acid precipitation and three M-protein preparations purified by cellulose chromatography were examined by SDS and polyacrylamide gel electrophoresis, and analyzed for amino acid composition and N-terminal amino acids. Fingerprinting (both tryptic and chymotryptic) was performed on the cellulose-purified preparations of M1, M12, and M29 proteins which showed these proteins to be structurally related. Trypsin produced maps with 37 to 42 peptides, whereas chymotrypsin digestion resulted in 8 to 12 peptides, depending on the M-type. Sequencing was performed on the M12 protein and tentative identification of nine N-terminal amino acids was made. Molecular weights of the cellulose and TCA-purified M-proteins were determined by SDS gel electrophoresis and chromatography on G-200 Sephadex, with comparable results, indicating molecular size of at least 23 000. The amino acid analyses of the 10 TCA-purified proteins followed the patterns established for M-proteins, with high concentrations of lysine, aspartic acid, glutamic acid, alanine, and leucine. All 10 proteins had L-alanine as their N-terminal amino acid. Evidence for a one way cross-reaction between type 1 and type 29 streptococci was also found.

A chemically defined medium (FMC; B. Terleckyj, N. P. Willett, and G. D. Shockman, Infect. Immun. 11 :649-655, 1975) was used to compare the growth and amino acid requirements of 16 strains of group B streptococci, consisting of both laboratory-passaged organisms and fresh clinical isolates from adult and neonatal infections. The 5 standard Lancefield immunizing strains of group B streptococci, 090 (Ia), H36B (Ib), A909 (Ic), 18RS21 (II), and D136C (III), had doubling times in FMC (28 to 36 min) similar to those observed in Todd-Hewitt glucose broth (24 to 30 min). Similar doubling times were obtained with 11 clinical isolates growing in Todd-Hewitt glucose broth and FMC. The optimum buffering capacity of FMC was provided by 0.06 M sodium phosphate, and 1% glucose gave maximum cell yield. The group B streptococci, with minor exceptions, were very homogeneous in their amino acid requirements under both aerobic and anaerobic growth conditions. Phenylalanine, tyrosine, tryptophan, glutamate, arginine, valine, leucine, lysine, methionine, isoleucine, cystine, and histidine were required by all 16 strains under both aerobic and anaerobic growth conditions. In addition, threonine was required by all strains under aerobic growth conditions, whereas only 9 strains required threonine under anaerobic conditions. Serine was required by only 3 type III fresh clinical isolates aerobically, but not anaerobically. A requirement for glycine varied from strain to strain, apparently influenced by the oxidation-reduction potential of the growth medium.

Type-specific M proteins were examined to determine whether their immunological specificities were also reflected by major chemical differences. Sixteen different type-specific protein preparations were employed, including two from non-M-typable strains. These M proteins, acid-extracted from whole cells, were purified by ammonium sulfate fractionation and column chromatography and were compared by immunodiffusion, electrophoretic, amino acid, and N-terminal amino acid analyses. Although the data did not reflect major chemical distinctiveness in the types examined, some interesting results evolved. Four important factors were observed to be shared by all M protein types examined: (i) glutamic acid was the most prevalent amino acid, (ii) amino acid molar ratios were similar, (iii) each had l -alanine as a single N-terminus, and (iv) purified peaks from the column chromatograms still showed heterogeneity while giving type-specific reactivity for multiple bands. Thus, whereas chemical typing is apparently unfeasible, the data indicate that these may be unique proteins, reflected especially in the finding of the same N-terminus amino acid in all strains investigated.

Are the conditions which are believed to be necessary for autolysis realized in infarction? A true acidity is known to be necessary in some critical cases for autolysis,1 that is, PH < 7, 0. Infarction was made by kidney vessel ligation. The CH was determined for the control blood and for that of the blood from the kidney after various periods of time elapsing after ligation.Again, in a guinea-pig liver, excised and frozen by CO2 within 50 seconds after excision, ground up and suspended in 0.9 per cent. NaCl solution and introduced into a Clark (W. M.) shaking hydrogen electrode CH gave PH 6, 5, the blood control giving 7, 2. After 35 minutes, PH = 6, 33 This rapid rise in CH is in harmony with the observations of Hopkins, Moore and Roaf, concerning the origin of lactic acid immediately after the death of the tissue. It is likewise compatible with the determinations which Taschiro4 has made on CO2 evolution after injury. The conclusion is justified that as far as reaction is concerned, the concentration o...

Bacterial mucopeptide is an integral part of bacterial cell walls and is therefore ubiquitous in our environment. An enhanced degree of humoral immunity has been detected not only in patients with acute rheumatic fever (ARF), with a known recent response to streptococci, but also in patients with adult and juvenile rheumatoid arthritis (RA and JRA). Our studies confirmed this association with ARF and JRA using a precipitin system as well as a radioimmunoassay to detect IgG anti-mucopeptide antibodies. In those with adult RA, either IgM or IgA rheumatoid factors or IgM or IgA antibodies specific for mucopeptide were responsible for the increased incidence of precipitins to mucopeptide in the RA patients detected in this and other studies. No differences in the specificities of the anti-mucopeptide antibodies were noted between the various patient populations as there were no lines of partial identity or nonidentity when examined by Ouchterlony double diffusion analyses. Additionally, no differences of anti-mucopeptide antibody were observed when the sera from these same patient populations were examined employing inhibition studies utilizing N-acetylglucosamine and rhamnose.

The concept "latent jaundice," i. e., a condition in which there is no visible discoloration of the skin and no excretion of bile pigment in the urine, owes its development in recent years to two laboratory methods for determining slight increases (over normal) of bile pigment in the blood serum. These are the methods of Van den Bergh and Fouchet. The former has been employed as a quantitative as well as a qualitative method. It has been shown that bilirubin is normally present in the blood in a concentration of approximately 0.3 to 0.5 part to 200,000; that visible jaundice occurs only when the bilirubin content in the blood is increased to 4 parts or more in 200,000, and that the excretion of bilirubin through the kidneys does not begin until the latter concentration has occurred. It is evident, then, that, in all cases of cholecystitis with visible jaundice, there