Test method development to evaluate hot,humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ?Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH‐adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ?Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Molecular characterization of the diversity of Clostridium chauvoei isolates collected from two bovine slaughterhouses: analysis of cross-contamination

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rate disease affecting mainly cattle and sheep. Carcasses of animals affected by the disease are the chief source of soil infection and considered as an ever-present threat to livestock health. A study was undertaken to examine the cross-contamination of C. chauvoei in two different bovine slaughterhouses using restriction endonuclease analysis (REA) and protein analysis. Samples from various sites of two different bovine slaughterhouses were screened and 34 isolates were identified by conventional techniques and 16S rRNA gene (rrs) sequencing. C. chauvoei were isolated from carcass, soil, and sewage from slaughterhouses examined. The isolates were differentiated using REA and whole-cell and excretory protein pattern analysis combined with numerical analysis and cluster formation. The α and β toxins produced by the strains were characterized. Our preliminary results suggest that REA combined with numerical analysis provides additional criteria and characteristic banding patterns for the study of the cross-contamination and characterization of C. chauvoei. The effects of temperature, oxygen tension, and enzymes on C. chauvoei hemolysin activity were also discussed. These microorganisms may be a potential contaminant of carcasses and widespread in soil of abattoir environments. The information of area-specific distribution of C. chauvoei strains and its toxin characteristics may give an efficient program in protecting cattle and other ruminants.     

Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of <Emphasis Type="Italic">Gracilaria vermiculophylla </Emphasis>(Ohmi) Papenfuss (Gracilariales,Rhodophyta)

Background  

Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae.     

Influence of hydrological conditions on the <Emphasis Type="Italic">Escherichia coli</Emphasis> population structure in the water of a creek on a rural watershed

Background  

Escherichia coli is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, although the aquatic environment could be a secondary habitat. The aim of this study was to investigate the effect of hydrological conditions on the structure of the E. coli population in the water of a creek on a small rural watershed in France composed of pasture and with human occupation.     

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest,after washing and simulated wholesale and retail distribution

Aims

To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0–25°C, dependent on season).

Methods and Results

Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near‐harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g^?1. Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure‐contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g^?1 after processing and retail storage. Manure‐contaminated leeks harboured 0·73–1·55 cfu E. coli O145 g^?1 after washing and storage. There was no cross‐contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure‐contaminated carrots. There were five post‐distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest.

Conclusions

Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce.

Significance and Impact

Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.     

Detection of aerosolized bacterial spores (<Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>) using free-flying honey bees (Hymenoptera: Apidae) as collectors

An aerosol cloud of Bacillus atrophaeus (previously B. subtilis variety niger) spores, an anthrax surrogate, was created in a large 0.4 ha (1 ac), bee-containing, open-mesh tent. Bees from a B. atrophaeus uncontaminated hive flying through the cloud adsorbed the spores in statistically significant quantities. After removal of the B. atrophaeus contaminated hive and introduction of another B. atrophaeus uncontaminated hive, the bees again were monitored for the next few days for B. atrophaeus spores. B. atrophaeus spores accumulated on the bees bodies following their exposure to the residual B. atrophaeus contamination in the tent. The spore loads on the bees quickly returned to background levels after the hives were removed from the contaminated tent area. It may therefore be practical to use honey bee colonies to monitor foraging areas for disease-causing spores.     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Background  

In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria.     

Septins localize to microtubules during nutritional limitation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane.     

A comparison of cecal colonization of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serotype Typhimurium in white leghorn chicks and <Emphasis Type="Italic">Salmonella</Emphasis>-resistant mice

Background  

Salmonellosis is one of the most important bacterial food borne illnesses worldwide. A major source of infection for humans is consumption of chicken or egg products that have been contaminated withSalmonella entericaserotype Typhimurium, however our knowledge regarding colonization and persistence factors in the chicken is small.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

A genetic algorithm-Bayesian network approach for the analysis of metabolomics and spectroscopic data: application to the rapid identification of Bacillus spores and classification of Bacillus species

Background  

The rapid identification of Bacillus spores and bacterial identification are paramount because of their implications in food poisoning, pathogenesis and their use as potential biowarfare agents. Many automated analytical techniques such as Curie-point pyrolysis mass spectrometry (Py-MS) have been used to identify bacterial spores giving use to large amounts of analytical data. This high number of features makes interpretation of the data extremely difficult We analysed Py-MS data from 36 different strains of aerobic endospore-forming bacteria encompassing seven different species. These bacteria were grown axenically on nutrient agar and vegetative biomass and spores were analyzed by Curie-point Py-MS.     

Detection of Clostridium tyrobutyricum spores using polyclonal antibodies and flow cytometry

Aims: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent‐labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Methods and Results: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10³ spores per 100 ml of milk, and results could be achieved in 2 h. Conclusions: FCM combined with fluorochrome‐conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. Significance and Impact of the Study: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.     

Planctomycetes dominate biofilms on surfaces of the kelp <Emphasis Type="Italic">Laminaria hyperborea</Emphasis>

Background  

Bacteria belonging to Planctomycetes display several unique morphological and genetic features and are found in a wide variety of habitats on earth. Their ecological roles in these habitats are still poorly understood. Planctomycetes have previously been detected throughout the year on surfaces of the kelp Laminaria hyperborea from southwestern Norway. We aimed to make a detailed investigation of the abundance and phylogenetic diversity of planctomycetes inhabiting these kelp surfaces.     

Clinical and biochemical changes in 53 Swedish dogs bitten by the European adder - <Emphasis Type="Italic">Vipera berus</Emphasis>

Background  

Every year many dogs in Sweden are bitten by Vipera berus, the only venomous viper in Sweden. This prospective study investigated clinical signs, some biochemical parameters, treatment, and progress of disease after snakebite in 53 dogs. Effects of treatment with and without glucocorticoids were evaluated.     

Polylepis woodland dynamics during the last 20,000 years

Aim

To determine the palaeoecological influences of climate change and human land use on the spatial distribution patterns of Polylepis woodlands in the Andes.

Location

Tropical Andes above 2,900 m between 2°S and 18°S of latitude.

Methods

Pollen and charcoal data were gathered from 13 Andean lake sediment records and were rescaled by the maximum value in each site. The rescaled pollen data were used to estimate a mean abundance and coefficient of variation to show woodland expansions/contractions and woodland fragmentation over the last 20,000 years. The rescaled charcoal was displayed as a 200‐year moving median using 500‐year bins to infer the influence of fire on woodland dynamics at landscape scale. Pollen and charcoal were compared with speleothem, clastic flux and archaeological data to assess the influence of moisture balance, glacial activity and human impact on the spatial distribution of Polylepis woodlands.

Results

Woodland expansion and fire were correlated with precipitation changes and glacier dynamics from c. 20 to 6 kcal bp (thousands of calibrated years before present). Charcoal abundances between 20 and 12 kcal bp were less common than from 12 kcal bp to modern. However, human‐induced fires were unlikely to be the main cause of a woodland decline centred at 11 kcal bp , as woodlands recovered from 10.5 to 9.5 kcal bp (about twofold increase). Charcoal peaks analogous to those that induced the woodland decline at 11 kcal bp were commonplace post‐9.5 kcal bp but did not trigger an equivalent woodland contraction. An increase in the coefficient of variation after c. 5.5 kcal bp suggests enhanced fragmentation and coincided with the shift from logistic to exponential growth of human populations. Over the last 1,000 years, Polylepis became hyper‐fragmented with over half of sites losing Polylepis from the record and with coefficients of variation paralleling those of glacial times.

Main conclusions

Polylepis woodlands formed naturally patchy woodlands, rather than a continuous vegetation belt, prior to human occupation in the Andes. The main factors controlling pre‐human woodland dynamics were precipitation and landscape heterogeneity. Human activity led to hyper‐fragmentation during the last c. 1,000 years.     

Molecular Detection of <Emphasis Type="Italic">Penicillium griseofulvum</Emphasis> as the Coastal Pollution Indicator

Molecular methods were carried out to detect Penicillium griseofulvum, a dominant species related to heavy metal pollution, which was screened from marine contaminated sediments. Based on differences in internal transcribed spacer (ITS) sequences of Penicillium genus and specific isoamyl alcohol oxidase (IAO) sequences, species-specific primers AS1/RS4 and IAO1/IAO2 of Penicillium griseofulvum were designed and synthesized which were then employed in optimized PCR systems. The detection sensitivities were compared through ordinary PCR and nested-PCR using two pairs of primers, respectively. Both primer pairs could exclusively amplify destined DNA fragment from contaminated environmental samples in our researches. As for primers AS1/RS4, the detection sensitivity for spores (pure spore DNA) could be 10 fg/μl and 10 spores, respectively, and the detection sensitivity for the sediments was 10² spores/0.25 g sediments. While the detection sensitivity of IAO1/IAO2 primers was lower than that of AS1/RS4. Despite the difference in detection sensitivity, it is feasible that the species-specific primers could be used as probes for the detection of environmental pollution dominant species, Penicillium griseofulvum, since the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.     

A longitudinal study of <Emphasis Type="Italic">Campylobacter</Emphasis> distribution in a turkey production chain

Background  

Campylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis. Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.