Toxin Titers Research Articles

Enhanced mucosal and systemic immune responses following intravaginal immunization with human papillomavirus 16 L1 virus-like particle vaccine in thermosensitive mucoadhesive delivery systems.

To develop more potent and convenient mucosal human papillomavirus (HPV) vaccines, we tested the effect of thermosensitive mucoadhesive vaginal vaccine delivery systems on the local and systemic antibody responses to HPV 16 L1 virus-like particles (VLP). HPV 16 L1 VLP expressed from recombinant baculovirus-infected Sf21 insect cells were delivered in phosphate-buffered saline (PBS) or thermosensitive mucoadhesive delivery systems, composed of poloxamers (Pol) and varying amounts of polyethylene oxide (PEO). Pol/PEO-based vaginal vaccine delivery systems existed in liquid form at room temperature, but gelled at 37 degrees C. The mucoadhesiveness of Pol/PEO-based delivery systems increased with PEO, but the formulations with PEO higher than 1.0% were too viscous to be administered into the vagina. Vaccine vehicles affected the vaginal and salivary immune responses to HPV 16 L1 VLP intravaginally administered into mice. At 42 days after the first intravaginal immunization of HPV 16 L1 VLP with cholera toxin, vaginal and salivary IgA titers were the highest in the group given in Pol/PEO 1.0% vehicle followed by Pol/PEO 0.4% and PBS vehicles. Intravaginal coadministration of HPV 16 L1 VLP and cholera toxin in Pol/PEO 1.0% showed 31- and 39-fold higher titers compared to the PBS-based HPV 16 L1 VLP groups administered by intravaginal and intramuscular routes, respectively. Following intravaginal administration, Pol/PEO 1.0%, but not Pol/PEO 0.4%, showed significantly higher HPV 16 L1 VLP-specific serum IgG titers as compared to the PBS vehicle. Our results indicate that the use of in situ-gelling vaginal vaccine delivery systems with increased mucoadhesiveness would be beneficial for more effective induction of mucosal and systemic immune responses to intravaginally administered HPV 16 L1 VLP vaccines.

The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces

Aims:Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research methods may necessitate storage of faecal specimens for long periods, followed by subsequent re-examination. This study investigated...

Immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine

Pasteurella multocida is an important bacterial pathogen of domestic rabbits. To evaluate the ability of a thiocyanate extract (PTE) of P. multocida to stimulate an immune response and protect against infection with P. multocida, rabbits were immunized subcutaneously or intranasally on Days 7, 21 and 35. Cholera toxin, a potent mucosal adjuvant, was included in one treatment group. Rabbits immunized subcutaneously (SC) or intranasally (IN) had significant increases in serum anti-PTE IgG but not IgA. In contrast, only rabbits immunized IN with PTE developed significant titers of nasal lavage anti-PTE IgA and cholera toxin significantly enhanced this response. In a second study rabbits were immunized via the drinking water with PTE incorporated into alginate microparticles on Days 7, 14 and 21. Mild increases in serum IgG were noted in rabbits immunized with PTE in microparticles, with or without cholera toxin, and this increase was significant ( P≤0.05) on Day 21 for rabbits receiving PTE and cholera toxin. Nasal lavage anti-PTE IgA was significantly ( P≤0.05) increased in rabbits immunized orally with PTE, with or without cholera toxin, in microparticles. This effect was not enhanced by cholera toxin. Rabbits orally immunized with PTE in microparticles had significantly fewer colony forming units of homologous P. multocida recovered from the lungs and nasopharynx following an intranasal challenge. These results demonstrate that PTE incorporated into alginate microparticles and administered orally is immunogenic and confers protective immunity.

Regulation of crystal protein biosynthesis by Bacillus thuringiensis: II. Effects of carbon and nitrogen sources

The regulation of crystal protein production in Bacillus thuringiensis 81 by sources of carbon and nitrogen was investigated. The highest titers of toxin were obtained on sucrose, lactose and inulin which also supported sporulation. Whey and molasses were also potential carbon substrates for toxin production. Other carbohydrates including glucose, glycerol, maltose, starch and dextrin yielded lower amounts of toxin. Nitrogen sources were found to exert the most profound controls. Peptone was the best organic nitrogen source, supporting optimum production and sporulation as well as high cell density. The formation of CryI and CryII toxin proteins was found to be differentially regulated by the inorganic nitrogenous compounds incorporated into the medium.

Bacillus cereus emetic toxin production in cooked rice

Three mesophilic strains of Bacillus cereus known to produce emetic toxin were used to model germination, growth and emetic toxin production in boiled rice cultures at incubation temperatures ranging from 8°C to 30°C. Minimum temperatures for germination and growth in boiled rice were found to be 15°C for all strains. Toxin production at 15°C was found to be significantly greater ( P<0·01; reciprocal toxin titre of 373±124) than at 20°C and 30°C (reciprocal toxin titres 112±37 and 123±41, respectively). Toxin production became detectable after 48 h incubation at 15°C, with a maximum titre reached by 96 h. At 20°C and 30°C, toxin production was detected at 24 h incubation, with a maximum titre reached by 72 h. Toxin production at 15°C was detectable at lower bacterial counts (6·2 log 10 cfu g −1), than with incubation at 20°C and 30°C (>7·0 log 10 cfu g −1). In this study, the lower temperature limit for germination and growth on solid laboratory medium was found to be 12°C for all strains, i.e. 3°C lower than that observed in boiled rice.

Growth and toxin production by Clostridium botulinum in fish (Mugil cephalus) and shrimp (Penaeus indicus) tissue homogenates stored under vacuum

Toxin production by Clostridium botulinum types A–E in mullet and shrimp tissue homogenates stored between 4 and 30°C under vacuum for 6 weeks was evaluated. Mullet and shrimp tissue homogenates were inoculated with C. botulinum spores, then packaged under vacuum and held at 4, 10, 15 and 30°C. At 30 and 15°C storage, growth and toxin production by types A–E resulted in both mullet and shrimp tissue homogenates. Only type E toxin was observed in the homogenates held at 4 and 10°C. The storage temperature and the growth substrate markedly influenced the toxin titres.

Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease.

Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3-9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (>/=1:4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid.

Immunogenicity of IRIV- versus alum-adjuvanted diphtheria and tetanus toxoid vaccines in influenza primed mice

The immunogenicity and protectivity of two different toxoid vaccines were compared in mice. In one formulation, toxoids (diphtheria or tetanus) were adsorbed to alumoxid, whereas in the other formulation the toxoids were crosslinked to immunopotentiating reconstituted influenza virosomes (IRIVs). A preimmunization with influenza antigens is necessary for a good anti-toxoid antibody response when the IRIV formulation was administered. After two immunizations with the IRIV- or alum-based vaccines, the IRIV-based formulation induced a higher humoral immune response than toxoids adsorbed to alum. Using an in vitro test, diphtheria toxin neutralizing antibodies were tested. Di-IRIV induced a significantly ( p=0.002) higher titer of diphtheria toxin neutralizing antibodies than Di-alum. Tetanus challenge experiments showed, that the IRIV-based tetanus vaccine induced a threefold higher titer of protective antibodies than the tetanus toxoid adsorbed to alum. Therefore, the IRIV-based formulations appeared to be superior to the alum-based vaccines in terms of immunogenicity and protectivity.

Edema disease as a model for systemic disease induced by Shiga toxin-producing E. coli.

Edema disease (ED) is a naturally occurring disease of weaned pigs caused by host adapted strains of E. coli that produce Shiga toxin (STEC). We determined the temporal and quantitative relationships between intestinal colonization by STEC, levels of Shiga toxin (Stx2e) in the gut, in the blood, and clinical manifestations of ED. Bacterial colonization (10(8) CFU/cm ileum) was highest 4 days post inoculation (pi) in animals that did not develop clinical disease and 6 days pi in animals with clinical signs of ED. The mean time for the development of clinical signs of ED was 6 days pi (range 4-10). Average peak titers of Stx2e in the ileum were 1:16,384 in asymptomatic animals and 1:32,768 in clinical animals. Titers of Stx2e in the feces reflected the toxin titers in the ileum but were lower. Intestinal titers of Stx2e and the density of bacterial colonization were predictive of clinical ED for a group of animals but not for individuals. Approximately 50% of the pigs that had Stx2e titers of > or = 1:4096 and a bacterial density of > or = 10(6) CFU/cm in their ileum, had clinical ED. Pigs that had intestinal Stx2e titers < 1:4096 were asymptomatic. Stx2e was detected in the red cell fraction of blood from some of the pigs with clinical ED and in some that were asymptomatic. Stx2e was not detected in the serum of any animals. ED may be a useful model for predicting the temporal and quantitative relationships between bacterial colonization, Stx levels in the gut and blood and systemic disease for STEC in other species.

Modeling the Dynamics of Adaptation to Transgenic Maize by European Corn Borer (Lepidoptera: Pyralidae)

A deterministic population dynamics model was modified to include single-locus, 2-allele genetics to simulate strategies for delaying resistance in the European corn borer, Ostrinia nubilalis (Hübner), population to transgenic maize. We evaluated seed mixtures of transgenic and nontransgenic maize, 2-row strips of non transgenic plants, adjacent but separate refuges of nontransgenic maize, and rotation of nontransgenic and transgenic maize. We studied how the choice of hybrid, planting time, and weather may influence the selection of a tactic for delaying resistance to transgenic maize. Our results indicate that separate refuges are superior to seed mixtures for delaying resistance. If a high toxin dose cannot be achieved and a small fraction of homozygous susceptible and heterozygous European corn borer neonates survive on transgenic maize, then resistance can develop in 10-33% of the time required under the assumption of a successful high dose that kills all heterozygous neonates. Planting 2-row strips may be as good as separate refuges in delaying resistance, but their adoption carries greater risk because of the uncertainty surrounding movement and survival of neonates. The use of transgenic hybrids is the same whether or not they express the toxin gene in silks and kernels of maize ears. Toxin titer decline must be studied because our simulations indicate that development of resistance may be greatly hastened by this process.

Effect of Isoleucine on Toxin Production by Clostridium difficile in a Defined Medium

Supplementation of a carbohydrate-free minimal medium with a high level (100 mM) of histidine, methionine, valine, isoleucine, proline and leucine, in particular isoleucine, markedly increased toxin production by Clostridium difficile VPI 10463. The effect of isoleucine was further examined. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decreased gradually. Amino acid analysis of the culture revealed that, at 100 mM isoleucine, consumption of isoleucine was remarkably increased. During the incubation period when toxin titers increased markedly but bacterial growth was declining, isoleucine, leucine and cysteine were taken up preferentially and alanine and cystathionine, which were not found at 1 mM isoleucine, were produced in large quantities. These findings suggest that isoleucine may play an important role in toxin production by C. difficile and that alanine and cystathionine production may be co-regulated with the toxin production in the absence of fermentable carbohydrates.

The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin.

Binding of the purified type C 7S (neurotoxin), 12S and 16S botulinum toxins to epithelial cells of ligated small intestine or colon of the guinea pig (in vivo test) and to pre-fixed gastrointestinal tissue sections (in vitro test) was analysed. The 16S toxin bound intensely to the microvilli of epithelial cells of the small intestine in both in vivo and in vitro tests, but did not bind to cells of the stomach or colon. The neurotoxin and 12S toxin did not bind to epithelial cells of the small intestine or to cells of the stomach or colon. Absorption of the toxins was assessed by determining the toxin titre in the sera of guinea pigs 6-8 h after the intra-intestinal administration of the toxins. When the 16S toxin [1 x 10(5) minimum lethal dose (MLD)] was injected, 200-660 MLD ml-1 was detected in the sera, whereas when the 12S toxin (2 x 10(5) MLD) or 7S toxin (2 x 10(5) MLD) was injected, little toxin activity was detected in the sera. Therefore, the haemagglutinin of type C 16S toxin is apparently very important in the binding and absorption of botulinum toxin in the small intestine.

Streptococcal Pyrogenic Exotoxin A, Streptolysin O, Exoenzymes, Serotype and Biotype Profiles of Streptococcus pyogenes Isolates from Patients with Toxic Shock Syndrome and other Severe Infections

The determination of protein M and T serotypes, biotypes and pyrogenic (erythrogenic) exotoxin A (SPE A), streptolysin O (SLO), streptokinase (SK), hyaluronidase (HA) and cysteine proteinase release by 212 S. pyogenes isolates from patients with severe invasive group A streptococcal (GAS) infections, among them 74 cases of streptococcal toxic shock syndrome (STSS) has been investigated. M1 or M3 serotypes were expressed by 25% of the isolates (53/212), whereas 59% (125/212) belonged to 15 other different serotypes and 16% (34/212) were untypeable. Of the 74 isolates from STSS patients, 42% (31/74) expressed M1 and to a lesser extent M3 serotypes versus 19% of the non STSS isolates (26/138). Among the ten different biotypes known, biotypes 1 and 3 were prevalent, particularly the former in the case of STSS isolates. SPE A was detectably produced by about 25% (54/212) of the strains. However, as high as 40.5% of the STSS isolates (30/74) versus 17.4% of non STSS isolates (24/138) released SPE A. Moreover, 67% of the SPE A producing strains were of serotype M1 or M3. SK and HA were released by 71% and 10% of the isolates respectively. All strains released SLO (4 to 256 HU/ml) and 85% cysteine proteinase. No relationship between toxin or enzyme titer and the type of disease or clinical origin of the strains was found. Culture supernatants of all isolates showed moderate to high lymphocyte transforming activity with index values ranging from 14.5 to 50.3 including those strains which did not release detectable amounts of SPE A suggesting that SPE C and other mitogenic factor(s) are released by the isolates investigated.

Production of diarrheal toxin by selected strains of Bacillus cereus

The production of diarrheal toxin by six selected strains of Bacillus cereus was monitored during growth at 32 °C, a temperature described as near-optimal for growth and toxin production. Toxic activity was measured in culture filtrates and cellular extracts sampled at three different times during growth. Two alternative methods, a cytotoxicity test on Chinese hamster ovary (CHO) cells and a commercial immunological test (BCET-RPLA, Oxoid) were used. Toxin titres were in agreement with epidemiological characteristics and toxicity demonstrated by using other systems in other examinations. A comparison of intra- and extracellular toxicities measured at the exponential and stationary growth phases showed that the toxin was essentially secreted during the exponential phase. For several strains, secretion peaked during the period from the middle exponential phase to the beginning of the stationary phase. There was no important overall increase of the toxicity during full and late stationary phase. The level was stable or even lower, thus indicating that diarrheal toxin production during stationary phase was small, if any, and that the toxin was unstable under these conditions. Statistical analysis of toxicities showed that the cytotoxicity test was correlated with the immunological test (significant at a 1% level). For routine determinations, a toxicologic laboratory may use any of the two methods, depending oft its facilities, the immunological test being relatively expensive.

Impact of Anaerobic Growth Conditions on Toxic Shock Syndrome Toxin‐I Production by Staphylococcus aureus

Objective: The impact of anaerobic growth conditions on the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) production was studied. Methods: Ten strains of S. aureus derived from patients with toxic shock syndrome (TSS), 10 isolates of S. aureus, and documented TSST-1-producing strains recovered from patients with either staphylococcal septicemia or staphylococcal nongenital abscesses were grown under aerobic and anaerobic conditions. The bacterial growth was measured using optical density (OD) determinations at 520 nm. The toxin production was assayed using the TS-RPLA latex agglutination test. Results: Both TSS and non-TSS strains of S. aureus grown under aerobic and anaerobic conditions exhibited comparable OD patterns of growth, and the levels of toxin production remained constant during the logarithmic phase. Toxin titers developed during the logarithmic growth phase and peaked after 24 h of incubation. When stationary-phase isolates grown initially under aerobic conditions were subjected to strict anaerobic conditions, subsequent toxin titers, compared with isolates grown in the continued presence of oxygen, were depressed 2-fold, peaking at a later time. Conclusions: TSST-1 production is diminished under continued anaerobic conditions.

Anti-tetanus toxin titers in sera of the women who gave births in 1985 and 1989 in Gipuzkoa (Basque Country, Spain).

14.9% (208/1400) of the women who gave births in 1985 and 1989 in Gipuzkoa (Basque Country, Spain), was considered to have 'protective' levels (> or = 0.01 IU/ml) of tetanus antitoxin as measured by the passive haemagglutination test. Prevalence of protective antibodies was correlated to the years when women were born: 54.7% (64/117) of those born after 1965 were considered to have 'protective' levels (> or = 0.01 IU/ml) of tetanus antitoxin in contrast to those born before 1956 [only 6.2% (21/338)]. This is considered to be due to the infant immunization campaign introduced in 1965 in the Basque Country. Thus, we conclude that the tetanus immunization campaigns in the Basque Country adult population is urgent.

Clostridium difficile Colitis: Correlation of CT findings with severity of clinical disease

Clinical records and abdominal CT scans from 64 patients with documented Clostridium difficile disease were reviewed to determine if any correlation existed between CT findings of colitis and severity of clinical disease. Clostridium difficile disease was documented with stool toxin titre levels and CT scans were performed within 3 days of stool sample. Clinical disease severity was estimated by tabulating the degree of fever, WBC count, frequency and duration of diarrhoea. Thirty-nine of 64 patients showed CT evidence of colitis of which 28/39 showed evidence of focal colitis and 11/39 had pancolitis. CT findings suggesting colitis included colonic wall thickening (39 patients), nodular mucosal thickening (11 patients), the 'accordion pattern' (3 patients), pericolonic oedema (27 patients) and ascites (10 patients). Twenty-five of 64 patients showed no CT evidence of colitis. The clinical severity of disease did not statistically differ (P < 0.05) between patients with CT evidence of colitis and those without colitis. The only CT finding that correlated with clinical severity of disease was nodular mucosal thickening which was found with significantly (P < 0.05) more frequency in patients with a WBC count > 11,000 mm3. CT changes with Cl. difficile disease correlate poorly with the clinical severity. This and negative findings do not exclude the disease.

Development and usefulness of the gelatin-particle-agglutination test for titration of antibodies against diphtheria, pertussis and tetanus toxins.

The gelatin-particle-agglutination (PA) test for titrating antibodies against diphtheria, pertussis and tetanus toxins was developed and used for assaying 65 sera from healthy children to assess the antitoxin acquisition in relation to the administration of adsorbed diphtheria-purified pertussis-tetanus (DPT) combined vaccine. The antitoxin titers obtained by the PA test and the conventional methods were correlated well; the correlation coefficient of the diphtheria antitoxin titers between the PA test and the cell culture method was 0.908, that of the tetanus antitoxin titers between the PA test and the passive hemagglutination test 0.968, and that of anti-pertussis toxin titers between the PA test and polystyrene-ball ELISA 0.885. The PA test was shown to be useful in both developed and developing countries, since it is simple to perform, sensitive and specific, and the three antitoxins can be titrated by the same procedure.

Neutralizing antibodies and immunoprotection against pertussis and tetanus obtained by use of a recombinant pertussis toxin-tetanus toxin fusion protein.

The currently available diphtheria-tetanus-whole-cell pertussis (DTP) vaccines are associated with a variety of problems, including undesirable side effects and inconsistent efficacy. These problems are probably related to the poor definition of such vaccines, especially with respect to the whole-cell component against pertussis. Ideal vaccines should include only immunoprotective antigens with no toxin activity. As an initial step towards obtaining a well-defined and simplified DTP vaccine, a pertussis toxin-tetanus toxin chimeric protein was constructed. A soluble form of the pertussis toxin S1 subunit was fused to the protective fragment C of tetanus toxin, and the recombinant hybrid protein was produced in Escherichia coli. The 75-kDa fusion protein (p75) was overexpressed as a soluble molecule and purified to near homogeneity by two consecutive chromatographic steps. Purified p75 retained its ability to bind to ganglioside GT1b, the receptor for tetanus toxin, and to be recognized by protective and neutralizing anti-pertussis toxin antibodies specific for conformational epitopes. When administered to mice, the hybrid protein was found to be nontoxic but immunogenic. In addition, it was capable of inducing strong protection against tetanus and some protection against pertussis, as well as eliciting a pertussis toxin-neutralizing antibody response. Although the levels of anti-pertussis toxin antibodies were rather low, neutralizing titers of the immunized mice correlated well with anti-pertussis toxin titers, indicating that protective epitopes are conserved in the recombinant protein.

Diversion colitis. A prospective study.

A prospective study of patients who had undergone fecal diversion was performed in order to determine the incidence of and to characterize better the condition known as "diversion colitis." A total of 53 patients were studied. All patients underwent endoscopic evaluation of the diverted large bowel. Evidence of colitis was found in 48 patients (91%). The colitis was mild in 52%, moderate in 44%, and severe in only 4%. Endoscopic findings included: contact irritation or bleeding, erythema, and mucosal nodularity. The proximal, "in continuity," colon was examined in 86% of patients with colostomies; none were found to have colitis. Biopsies were taken of the diverted segment in 94% of patients with colitis and from the "in continuity" colon in 78% of patients with colostomies. Similar histologic findings were noted on these biopsies and included: mild chronic inflammation, lymphoid nodules, and crypt architectural changes. With the exception of lymphoid nodules, which were seen more frequently in the inflamed diverted colon (P = 0.035), there was no significant difference in the incidence of the various histologic changes when the biopsies from the diverted and "in continuity" large bowel were compared. Rectal washings and stool samples were sent for bacterial cultures, ova, and parasite analysis, and C. Diff. toxin titers in the majority of patients; all but 1 were negative. Symptoms relating to the diverted bowel were elicited in only 3 patients (6%). Stomal closure was carried in 70% of patients. Postclosure endoscopy in 21 patients revealed full resolution of the colitis in all. Diversion colitis occurs in almost all diverted patients. It uniformly resolves following stomal closure.(ABSTRACT TRUNCATED AT 250 WORDS)