Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens

Cost effective control of avian diseases and food borne pathogens remains a high priority for all sectors of the poultry industry with cleansing and disinfection, vaccination and competitive exclusion approaches being used widely. Previous studies showed that Bacillus subtilis PY79(hr) was an effective competitive exclusion agent for use in poultry to control avian pathogenic Escherichia coli serotype O78:K80. Here we report experiments that were undertaken to test the efficacy of B. subtilis PY79(hr) in the control of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. To do this, 1-day-old and 20-day-old specific pathogen free (SPF) chicks were dosed with a suspension of B. subtilis spores prior to challenge with S. Enteritidis (S1400) and C. perfringens, respectively. For both challenge models, a single oral inoculum of 1 x 10(9) spores given 24 h prior to challenge was sufficient to suppress colonisation and persistence of both S. Enteritidis and C perfringens. In particular, the faecal shedding of S. Enteritidis, as measured by a semi-quantitative cloacal swabbing technique, was reduced significantly for the 36 days duration of the experiment. B. subtilis persisted in the intestine although with decreasing numbers over the same period. These data add further evidence that B. subtilis spores may be effective agents in the control of avian diseases and food borne pathogens.

Premature germination of resting spores as a means of Protecting brassica crops from Plasmodiophora brassicae Wor., (clubroot)

Premature germination of resting spores as a means of protecting brassica crops from Plasmodiphora brassicae Wor., (Clubroot). Crop Protection. Clubroot disease causes substantial yield and quality losses in broadacre oil seed and intensive vegetable brassica crops worldwide. The causal microbe Plasmodiophora brassicae Wor., perennates as soil-borne dormant resting spores. Their germination is triggered by exudates from host roots. A valuable addition to sustainable integrated control strategies could be developed by identifying and synthesising the molecules responsible for stimulating resting spore germination. This paper reports experiments in which stimulatory exudates were collected from brassica roots following exposure to infective stages of P. brassicae. Analyses identified a germination signalling molecule of circa 1 kDa formed of glucose sub-units. Mass spectral analyses showed this to be a complex hexasaccharide carbohydrate with structural similarities to the components of plant cell walls. This is the first report of a host generated hexasaccharide which is capable of stimulating the germination of resting spores of P. brassicae. The implications for environmentally benign control of clubroot are discussed briefly.

Indoor and outdoor atmospheric fungal spores in the So Paulo metropolitan area (Brazil): species and numeric concentrations

The aim of this study was to estimate the indoor and outdoor concentrations of fungal spores in the Metropolitan Area of Sao Paulo (MASP), collected at different sites in winter/spring and summer seasons. The techniques adopted included cultivation (samples collected with impactors) and microscopic enumeration (samples collected with impingers). The overall results showed total concentrations of fungal spores as high as 36,000 per cubic meter, with a large proportion of non culturable spores (around 91% of the total). Penicillium sp. and Aspergillus sp. were the dominant species both indoors and outdoors, in all seasons tested, occurring in more than 30% of homes at very high concentrations of culturable airborne fungi [colony forming units(CFU) m(-3)]. There was no significant difference between indoor and outdoor concentrations. The total fungal spore concentration found in winter was 19% higher than that in summer. Heat and humidity were the main factors affecting fungal growth; however, a non-linear response to these factors was found. Thus, temperatures below 16A degrees C and above 25A degrees C caused a reduction in the concentration (CFU m(-3)) of airborne fungi, which fits with MASP climatalogy. The same pattern was observed for humidity, although not as clearly as with temperature given the usual high relative humidity (above 70%) in the study area. These results are relevant for public health interventions that aim to reduce respiratory morbidity among susceptible populations.

IN-VITRO INHIBITION OF SPORES GERMINATION OF ALTERNARIA-SOLANI BY 3 FUNGICIDES

In vitro inhibition of the of spores germination of Alternaria solani by iprodione, chlorothalonil, and anilazine at different dosages was studied. The highest concentration of active ingredient studied for each fungicide was equivalent to that recommended for the control of the early blight, under field conditions: 0.75; 1.80 and 1.44 g, respectively, of iprodione, chlorothalonil and anilazine per litre of water. A series of two-fold diluitions of each original concentration was studied in additional nine experiments. Eah of the three fungicides showed total in vitro spore inhibition at the highest rate, at six hours of incubation. At nine hours, only analazine mantained its full inhibition activity. The inhibition activity of iprodione decreased suddenly after 1/2 dilution, so that at the 1/8 dilution a total loss of inhibitory activity was observed. Chlorothalonil showed a progressive and slighter decrease of its activity as the dilution rate increased.Analizine showed a high inhibitory activity at higher dilutions, without any loss up to 1/128 dilution. Even at 1/512 dilution, its activity was so high that only 20% of spore germination was observed at six or nine hours of incubation.

Study of the presence of the spores of Clostridium botulinum in honey in Brazil

The isolation of Clostridium botulinum from honey samples is described. Botulism is characterized as an intoxication provoked by ingestion of contaminated foods with this toxin. Infant botulism happens by the ingestion of spores of C. botulinum together with food that in special conditions of the intestinal tract, such as those present in babies of less than 1 year old, will allow the germination and colonization of the intestine with production and absorption of botulinic toxin. The samples were subjected to dilution and to a thermal shock and cultivated in modified CMM (Difco). Cultures were subjected to Gram smears and toxicity tests in mice. The toxic cultures were purified in RFCA (Oxoid) plates and incubated in anaerobic jars. Positive samples were typed using the mouse assay neutralization test. From the 85 honey samples analyzed, six were positive for C. botulinum (7.06%), and identified as producers of type A, B, and D toxins.

Solubilization of iron phosphate by free or immobilized spores and pellets of Aspergillus niger

The production of pellets by Aspergillus niger and the FePO 4 (FeP) solubilization through the use of free or immobilized spores and pellets were studied. The media Sabouraud, MS-FeP, MS-K 2HPO 4, Czapek and Malt were used for pellets yield. Enhanced growth and pellets production were found in the Sabouraud and MS-FeP than those produced by the other media. Pellets produced in Sabouraud were larger in size and formed well-defined spheres than those produced in MS-FeP. Pellets amounts varying from 2 to 8 mg mL, -1 were inoculated in the MS-FeP medium. The greatest quantity of solubilized FeP was found when 6 mg of pellets mL, -1 were used. While the free spores were the worst form used for FeP solubilization in culture medium, free pellets allowed for the production of the greatest quantities of soluble phosphate, even after repeated use of the pellets. In the soil, pellets solubilized similar quantities of FeP compared to the immobilized A. niger spores and can be used with advantage since they are easily produced. © 2010 Academic Journals Inc.

Antimicrobial effects of ozonated water on the sanitization of dental instruments contaminated with E. coli, S. aureus, C. albicans, or the spores of B. atrophaeus

Objectives: Ozone has been used as an alternative method for the decontamination of water, food, equipment and instruments. The objective of this study was to evaluate the antimicrobial effects of ozonated water on the sanitization of dental instruments that were contaminated by Escherichia coli, Staphylococcus aureus, Candida albicans and the spores of Bacillus atrophaeus. Methods: A total of one hundred and twenty standardized samples of diamond dental burs were experimentally contaminated with E. coli (ATCC 25922), S. aureus (ATCC 6538) and C. albicans (ATCC 18804) and the spores of B. atrophaeus (ATCC 6633) for 30min. After the contamination, the samples were exposed to ozonated water (10mg/L O3) for 10 or 30min. The control group was composed of samples that were exposed to distilled water for 30min. After the exposure to the ozonated water, 0.1mL aliquots were seeded onto BHI agar to count the colony-forming units per milliliter (CFU/mL) of E. coli, S. aureus, and B. atrophaeus. Sabouraud dextrose agar was used to count the CFU/mL of C. albicans. The results were subjected to an analysis of variance and the Tukey test. Results: For all of the microorganisms studied, the ozonated water reduced the number of CFU/mL after 10 and 30. min of sanitization, and this microbial reduction was dependent on the duration of the exposure to the ozonated water. E. coli exhibited the greatest reduction in CFU/mL (2.72-3.78. log) followed by S. aureus (2.14-3.19. log), C. albicans (1.44-2.14. log) and the spores of B. atrophaeus (1.01-1.98. log). Conclusion: The ozonated water was effective in reducing the CFU of E. coli, S. aureus, C. albicans and B. atrophaeus spores, suggesting that ozonated water can be used for the sanitization of dental instruments. © 2012 King Saud Bin Abdulaziz University for Health Sciences.

Bacillus atrophaeus inactivated spores as a potential adjuvant for veterinary rabies vaccine

Rabies is a viral encephalitis, nearly always fatal, but preventable through vaccines. Rabid animal bite is the prime transmission act, while veterinary vaccination is one of the best strategies for rabies general prevention. Aluminum compounds and saponin are the commercial adjuvants used for this vaccine nowadays. Nevertheless, aluminum compounds can provoke undesired side effects and saponin has a narrow activity range without toxicity. B. atrophaeus inactivated spores (BAIS), with or without saponin, were then used as an alternative to boost the inactivated rabies virus response. BAIS was as effective as saponin in augmenting antibody titers, but combination of both adjuvants doubled the titers raised by them individually. The combined adjuvant formulation maintained viability for 21 months when stored at 4-8 degrees C. Overall, BAIS was demonstrated as a viable alternative to commercial adjuvants, while its combination with saponin resulted in even higher vaccine potency with good stability. (C) 2012 Elsevier Ltd. All rights reserved.

Investigating the Role of Small, Acid-Soluble Spore Proteins (SASPs) in the Resistance of Clostridium Perfringens Spores to Heat.

BACKGROUND: Clostridium perfringens type A food poisoning is caused by enterotoxigenic C. perfringens type A isolates that typically possess high spore heat-resistance. The molecular basis for C. perfringens spore heat-resistance remains unknown. In the current study, we investigated the role of small, acid-soluble spore proteins (SASPs) in heat-resistance of spores produced by C. perfringens food poisoning isolates. RESULTS: Our current study demonstrated the presence of all three SASP-encoding genes (ssp1, 2 and 3) in five surveyed C. perfringens clinical food poisoning isolates. beta-Glucuronidase assay showed that these ssp genes are expressed specifically during sporulation. Consistent with these expression results, our study also demonstrated the production of SASPs by C. perfringens food poisoning isolates. When the heat sensitivities of spores produced by a ssp3 knock-out mutant of a C. perfringens food poisoning isolate was compared with that of spores of the wild-type strain, spores of the ssp3 mutant were found to exhibit a lower decimal reduction value (D value) at 100 degrees C than exhibited by the spores of wild-type strain. This effect was restored by complementing the ssp3 mutant with a recombinant plasmid carrying wild-type ssp3, suggesting that the observed differences in D values between spores of wild-type versus ssp3 mutant was due to the specific inactivation of ssp3. Furthermore, our DNA protection assay demonstrated that C. perfringens SASPs can protect DNA from DNase I digestion. CONCLUSION: The results from our current study provide evidences that SASPs produced by C. perfringens food poisoning isolates play a role in protecting their spores from heat-damage, which is highly significant and relevant from a food safety perspective. Further detailed studies on mechanism of action of SASPs from C. perfringens should help in understanding the mechanism of protection of C. perfringens spores from heat-damage.

The characterization of residential fungal spores and the relationship with housing characteristics in the Houston area

A number of indoor environmental factors, including bioaerosol or aeroallergen concentrations have been identified as exacerbators for asthma and allergenic conditions of the respiratory system. People generally spend 90% to 95% of their time indoors. Therefore, understanding the environmental factors that affect the presence of aeroallergens indoors as well as outdoors is important in determining their health impact, and in identifying potential intervention methods. This study aimed to assess the relationship between indoor airborne fungal spore concentrations and indoor surface mold levels, indoor versus outdoor airborne fungal spore concentrations and the effect of previous as well as current water intrusion. Also, the association between airborne concentration of indoor fungal spores and surface mold levels and the age of the housing structure were examined. Further, the correlation between indoor concentrations of certain species was determined as well. ^ Air and surface fungal measurements and related information were obtained from a Houston-area data set compiled from visits to homes filing insurance claims. During the sampling visit these complaint homes exhibited either visible mold or a combination of visible mold and water intrusion problems. These data were examined to assess the relationships between the independent and dependent variables using simple linear regression analysis, and independent t-tests. To examine the correlation between indoor concentrations of certain species, Spearman correlation coefficients were used. ^ There were 126 houses sampled, with spring, n=43 (34.1%), and winter, n=42 (33.3%), representing the seasons with the most samples. The summer sample illustrated the highest geometric mean concentration of fungal spores, GM=5,816.5 relative to winter, fall and spring (GM=1,743.4, GM=3,683.5 and GM=2,507.4, respectively). In all seasons, greater concentrations of fungal spores were observed during the cloudy weather conditions. ^ The results indicated no statistically significant association between outdoor total airborne fungal spore concentration and total living room airborne fungal spore concentration (β = 0.095, p = 0.491). Second, living room surface mold levels were not associated with living room airborne fungal spore concentration, (β= 0.011, p = 0.669). Third, houses with and without previous water intrusion did not differ significantly with respect to either living room (t(111) = 0.710, p = 0.528) or bedroom (t(111) =1.673, p = 0.162) airborne fungal spore concentrations. Likewise houses with and without current water intrusion did not differ significantly with respect to living room (t(109)=0.716, p = 0.476) or bedroom (t(109) = 1.035, p = 0.304) airborne fungal spore concentration. Fourth, houses with and without current water intrusion did not differ significantly with respect to living room (χ 2 (5) = 5.61, p = 0.346), or bedroom (χ 2 (5) = 1.80, p = 0.875) surface mold levels. Fifth, the age of the house structure did not predict living room (β = 0.023, p = 0.102) and bedroom (β = 0.023, p = 0.065) surface mold levels nor living room (β = 0.002, p = 0.131) and bedroom (β = 0.001, p = 0.650) fungal spore airborne concentration. Sixth, in houses with visually observed mold growth there was statistically significant differences between the mean living room concentrations and mean outdoor concentrations for Cladosporium (t (107) = 11.73, p < 0.0001), Stachybotrys (t (106)=2.288, p = 0.024, and Nigrosporia (t (102) = 2.267, p = 0.025). Finally, there was a significant correlation between several living room fungal species pairs, namely, Cladosporium and Stachybotrys (r = 0.373, p <0.01, n=65), Curvularia and Aspergillus/Penicillium (r = 0.205, p < 0.05, n= 111)), Curvularia and Stachybotrys (r = 0.205, p < 0.05, n=111), Nigrospora and Chaetomium (r = 0.254, p < 0.01, n=105) and Stachybotrys and Nigrospora (r = 0.269, p < 0.01, n=105). ^ This study has demonstrated several positive findings, i.e., significant pairwise correlations of concentrations of several fungal species in living room air, and significant differences between indoor and outdoor concentrations of three fungal species in homes with visible mold. No association was observed between indoor and outdoor fungal spore concentrations. Neither living room nor bedroom airborne spore concentrations and surface mold levels were related to the age of the house or to water intrusion, either previous or current. Therefore, these findings suggest the need for evaluating additional parameters, as well as combinations of factors such as humidity, temperature, age of structure, ventilation, and room size to better understand the determinants of airborne fungal spore concentrations and surface mold levels in homes. ^

Evaluation of elevator shafts as a pathway for fungal spores and particles to enter a hospital housing immuno-compromised patients

Background. It is estimated that hospitals spend between 28 and 33 billion dollars per year as a result of hospital-acquired infections. (Scott, 2009) The costs continue to rise despite the guidance and controls provided by hospital infection control staff to reduce patient exposures to fungal spores and other infectious agents. With all processes and controls in place, the vented elevator shaft represents an unprotected opening from the top of the building to the lower floors. The hypothesis for this prospective study is that there is a positive correlation between the number of Penicillium/Aspergillus-like spores, Cladosporium, ascospores, basidiospores in spores/m3 as individual spore categories found in the hoistway vent of an elevator shaft and the levels of the same spores, sampled near-simultaneously in the outdoor intake of the elevator shaft. Specific aims of this study include determining if external Penicillium/Aspergillus-like spores are entering the healthcare facility via the elevator shaft and hoistway vents. Additional aims include determining levels of Penicillium/Aspergillus-like spores outdoors, in the elevator shafts, and indoors in areas possibly affected by elevator shaft air; and, finally, to evaluate whether any effect is observed due to the installation of a hoistway vent damper, installed serendipitously during this study. ^ Methods. Between April 2010 and September 2010, a total of 3,521 air samples were collected, including 363 spore trap samples analyzed microscopically for seven spore types, and polymerase chain reaction analyses on 254 air samples. 2178 particle count measurements, 363 temperature readings and 363 relative humidity readings were also obtained from 7 different locations potentially related to the path of air travel inside and near a centrally-located and representative elevator shaft. ^ Results. Mean Penicillium/Aspergillus-like spore values were higher outside the building (530 spores/m3 of air) than inside the hoistway (22.8 spores/m3) during the six month study. Mean values inside the hospital were lower than outside throughout the study, ranging from 15 to 73 spores/m3 of air. Mean Penicillium/Aspergillus-like spore counts inside the hoistway decreased from 40.1 spores/m3 of air to 9 spores/m3 of air following the installation of a back draft damper between the outside air and the elevator shaft. Comparison of samples collected outside the building and inside the hoistway vent prior to installing the damper indicated a strong positive correlation (Spearman's Rho=0.8008, p=0.0001). The similar comparison following the damper installation indicated a moderate non-significant inverse correlation (Spearman's rho = −0.2795, p=0.1347). ^ Conclusion. Elevator shafts are one pathway for mold spores to enter a healthcare facility. A significant correlation was detected between spores and particle counts inside the hoistway and outside prior to changes in the ventilation system. The insertion of the back draft damper appeared to lower the spore counts inside the hoistway and inside the building. The mold spore counts in air outside the study building were higher in the period following the damper installation while the levels inside the hoistway and hospital decreased. Cladosporium and Penicillium/Aspergillus -like spores provided a method for evaluating indoor air quality as a natural tracer from outside the building to inside the building. Ascospores and basidiospores were not a valuable tracer due to low levels of detection during this study. ^ Installation of a back draft damper provides additional protection for the indoor environment of a hospital or healthcare facility, including in particular patients who may be immunocompromised. Current design standards and references do not require the installation of a back draft damper, but evaluation of adding language to relevant building codes should be considered. The data indicate a reduction in levels of Penicillium/Aspergillus -like spores, particle counts and a reduction in relative humidity inside of the elevator shaft after damper installation.^

Compound specific stable isotopes, BIT, pollen and spores of Miocene to Pliocene sediments of ODP Hole 175-1085A

Pollen and stable carbon (d13C) and hydrogen (dD) isotope ratios of terrestrial plant wax from the South Atlantic sediment core, ODP Site 1085, is used to reconstruct Miocene to Pliocene changes of vegetation and rainfall regime of western southern Africa. Our results reveal changes in the relative amount of precipitation and indicate a shift of the main moisture source from the Atlantic to the Indian Ocean during the onset of a major aridification 8 Ma ago. We emphasise the importance of declining precipitation during the expansion of C4 and CAM (mainly succulent) vegetation in South Africa. We suggest that the C4 plant expansion resulted from an increased equator-pole temperature gradient caused by the initiation of strong Atlantic Meridional Overturning Circulation following the shoaling of the Central American Seaway during the Late Miocene.