Development of a novel multiplexed qPCR and Pyrosequencing method for the detection of human pathogenic yersiniae.

The purpose of this study was to develop a novel and robust molecular assay for the detection of human pathogenic yersiniae (i.e. Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis) in complex food samples. The assay combines multiplexed real-time PCR (qPCR) and Pyrosequencing for detecting and differentiating human pathogenic yersiniae with high confidence through sequence based confirmation. The assay demonstrated 100% specificity and inclusivity when tested against a panel of 14 Y. enterocolitica, 22 Y. pestis, 24 Y. pseudotuberculosis and a diverse selection of 17 other non-Yersinia bacteria. Pyrosequencing reads ranged from 28 to 40bp in length and had 94-100% sequence identity to the correct species in the GenBank nr database. Microbial enrichments of 48 ready-to-eat foods collected in the Greater Toronto Area from March 2014 to May 2014, including 46 fresh sprout and 2 salad products, were then tested using the assay. All samples were negative for Y. pestis and Y. pseudotuberculosis. Both salads (n=2) and 35% of sprout products (n=46) including 7.1% of alfalfa sprouts (n=14), 81% of bean sprouts (n=16), 12% of mixed sprouts (n=8) tested positive for Y. enterocolitica which was not detected in broccoli sprouts (n=5), onion sprouts (n=1), and pea sprouts (n=2). Cycle thresholds (Ct) of positive samples for Y. enterocolitica were between 23.0 and 37.9 suggesting post enrichment concentrations of approximately 1×102 to 1×106Y. enterocolitica per 1mL of enriched broth. An internal amplification control which was coamplified with targets revealed PCR inhibition in five samples which was resolved following a one in ten dilution. Pyrosequencing of qPCR amplicons suggests monoclonality and revealed a single nucleotide polymorphism that is present in Y. enterocolitica biotype 1A suggesting low pathogenicity of the detected strains. This study is the first to combine Pyrosequencing and qPCR for the detection of human pathogenic yersiniae and is applicable to a broad range of complex samples including ready-to-eat food samples.

Heat and desiccation are the predominant factors affecting inactivation of Bacillus licheniformis and Bacillus thuringiensis spores during simulated composting.

AIMS: The suitability of composting for disposal of livestock mortalities due to Bacillus anthracis was assessed by measuring viability of surrogate spores from two strains each of Bacillus licheniformis and Bacillus thuringiensis after a heating cycle modelled on a cattle composting study. METHODS AND RESULTS: Sporulation was attempted from 10 to 37°C, but poor yields at lower temperatures resulted in 25, 30 and 37°C being selected to generate sufficient spores (8 log10  CFU ml(-1) ) for experiments. Spores were inoculated into 3 g autoclaved dried-ground compost rehydrated with 6 ml water or silica beads in a factorial design for each strain, sporulation temperature, matrix and sampling day (0, 25, 50, 100, 150). Maximum incubation temperature was 62°C, but spores were maintained at ≥55°C for 78 of 150 days. Although significant differences existed among Bacillus strains and sporulation temperatures, numbers of viable spores after 150 days averaged 1·3 log10  CFU g(-1) , a 5·2 log10 reduction from day 0. CONCLUSIONS: Spore inactivation was likely due to heat and desiccation as matrices were autoclaved prior to incubation, negating impacts of microflora. SIGNIFICANCE AND IMPACT OF STUDY: Results support composting for disposal of anthrax mortalities, provided long-term thermophillic heating is achieved. Due to limited sporulation at 10°C, livestock mortalities from anthrax at this or lower ambient temperatures would likely be of lower risk for disease transmission.

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 × 10(1) and 1·3 × 10(2)  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.

Attenuation of an avian pathogenic Escherichia coli strain due to a mutation in the rpsL gene.

The diseases caused by pathogenic Escherichia coli constitute a major economic loss to the poultry industry. The development of a live oral E. coli vaccine to prevent or reduce diseases in poultry had been the objective of our work. Four spontaneous streptomycin-dependent (str-dependent) mutants were generated from a virulent avian strain that contains a mutation in the fur region of the chromosome. Genetic analysis of the mutants indicated that the str-dependent phenotype was due to a base change of C --> T at base 272 in the rpsL gene. The mutants were tested for attenuation using the day-old chick model. Day-old birds, in groups of 20, were either challenged with 10(6) colony-forming units (CFU) of the str-dependent mutant, the parent strain (containing the fur mutation), or the wild-type strain without the fur mutation. The parent strain and the wild-type strain were highly virulent, and 80% or more of the birds died. None of the birds challenged with the str-dependent mutants died, indicating attenuation of the mutants. The protective effect of the mutant as a live vaccine against the challenge with 10(6) CFU of the wild-type strain EC317 was investigated. Vaccination by both aerosol (day 1) and oral (days 14 and 28) routes using 10(8) CFU of the str-dependent mutant (EC1598) had no effect on the occurrence of cellulitis in the birds. Two vaccinations given as aerosol on day 1 and given orally on day 14 also had no significant effect on the occurrence of systemic lesions. Three immunizations on days 1, 14, and 28 resulted in a significant reduction in the number of birds with systemic lesions. Antibody titers prior to challenge were not predictive of outcome of challenge.

Chemical composition of endotoxins produced by Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme.

The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate.

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A2 but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A2 were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.

Interactions between Fusobacterium necrophorum hemolysin, erythrocytes and erythrocyte membranes.

The interactions between the hemolysin of Fusobacterium necrophorum subsp. necrophorum, erythrocytes and erythrocyte membranes were studied as an attempt to determine the initial characteristics leading to hemolysis. The spectrum of erythrocyte sensitivity indicated that horse, dog and mouse erythrocytes were highly sensitive whereas those of cattle, sheep, goat and chicken were insensitive to the hemolysin. Binding of hemolysin to horse and dog erythrocytes or their ghosts was more pronounced than to those of cattle and sheep as detected by a decrease of hemolytic activity from hemolysin preparations. The kinetics of hemolysis revealed that lysis is preceded by a prelytic phase characterized by binding of hemolysin to erythrocytes. Treatment of horse erythrocytes with hemolysin at various temperatures prior to incubation at 37 degrees C also revealed that this binding prelytic phase is temperature independent. This was followed by a temperature dependent lytic stage since erythrocytes pretreated with hemolysin and incubated at 4 degrees C showed no hemolysis. An inverse relation was found between erythrocyte concentration and hemolytic activity suggesting a multiple-hit mechanism of hemolysis.

The effect of Bcg gene on antigen presentation of spleen adherent cells and peritoneal macrophages from Mycobacterium bovis BCG-infected Bcg(s) and Bcg(r) mice.

To determine the role of the Bcg gene in the resistance and susceptibility of BCG-infected C57BL/6 (Bcg(s)) and its Bcg(r) congenic mice, the antigen presenting ability of spleen adherent cells and peritoneal macrophages, delayed type hypersensitivity (DTH) and lymphocyte blastogenic responses were investigated. The results obtained indicate that the DTH and lymphocyte blastogenic responses in Bcg(r) congenic mice were higher than in the Bcg(s) mice. Stimulation of spleen adherent cells with live Mycobacterium bovis BCG or PPD-BCG resulted in a higher antigen presenting ability in Bcg(r) than in Bcg(s) mice. However, comparatively low responses were associated with M. avium stimulation, with those in Bcg(r) being higher than in Bcg(s). I-A expression was also comparatively higher in Bcg(r) than in Bcg(s) mice. This study demonstrates that the Bcg gene seems to exhibit some effect on the antigen presenting ability of macrophages in immune responses of the congenic mice.

The effects of physical and chemical agents on the secretion and stability of a Fusobacterium necrophorum hemolysin.

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.

Establishment of Bcgr congenic mice and their susceptibility/resistance to mycobacterial infection.

Bcg congenic mice were developed by using C57BL/6 and DBA/2 strains of mice as progenitors. They were obtained by introgressively backcrossing the Bcgr marker of DBA/2 onto C57BL/6. After twenty successive backcrossings, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Bcgr congenic mice. The results of immunogenic and genetic markers coupled with those of an mixed lymphocyte reaction, all confirmed that the newly developed mice were highly congenic. These congenic mice were found to be resistant to in vivo infections by Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis BCG.

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Immune responsiveness in Mycobacterium avium-infected mice: changes in the proportion of T cell subsets and antibody production during the course of infection.

The C57Bl/6 susceptible (Bcgs) and its resistant (Bcgr) congenic mouse, previously developed by retrogressive backcrossing, were infected with 1 x 10(6) colony-forming units (CFU) of Mycobacterium avium and bacterial growth and their immune responses during the early and prolonged periods of infection were examined. There was a high proliferation in the liver and spleen of Bcgs mice, whereas no proliferation was observed in the Bcgr mice. Similarly, the sizes and weights of these organs were much higher than those of their Bcgr counterparts. The size and number of granulomas in Bcgs were also found to be higher than those of Bcgr. The CD3+ and CD4+ subsets increased dramatically in both mice during the early stage of infection. However, in the later phase of the infection, these populations decreased dramatically in Bcgs mice, but not in Bcgr mice, resulting in a depression in cell-mediated immune responses. No significant decrease in cell-mediated immune responses was observed in Bcgr mice even after prolonged infection. ELISA was performed to determine the antibody levels in both mice, and it was found that serum IgG and IgM levels in Bcgs were comparatively higher than those in Bcgr mice throughout the period of infection. The Bcg gene therefore may have an important role in the maintenance of resistance not only in the early phase but also in the later phase of Myco. avium infection.

Studies on the factors affecting the hemolytic activity of Fusobacterium necrophorum.

The in vitro activity of the hemolysin of Fusobacterium necrophorum was determined using the hemolysis of horse erythrocytes as an assay. The effects of medium composition and pH on hemolysin production were investigated. Calf serum and casitone stimulated a comparatively higher hemolytic activity in F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively. However, sugars, such as glucose, galactose and fructose were inhibitors of hemolytic activity. The spectrum of erythrocyte sensitivity to the hemolysin indicated that horse and quail erythrocytes were more sensitive to the hemolysin of both F. necrophorum subsp. necrophorum and subsp. funduliforme, than were cat, dog, rabbit, pigeon and human erythrocytes. Cat erythrocytes were however insensitive to the hemolysin of subsp. funduliforme. Cattle, sheep and chicken erythrocytes were insensitive to the hemolysin of the two subspecies. Medium pH near neutral were more effective in enhancing hemolytic activity, and hemolytic activity was positively correlated with growth. In general, F. necrophorum subsp. necrophorum was more hemolytic than subsp. funduliforme.

Comparison of extracellular enzymes of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme.

A total of 10 strains each of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were tested for the production of 13 extracellular enzymes. DNase, alkaline phosphatase, and lipase were predominantly associated with all the strains of F. necrophorum subsp. necrophorum, with DNase not detected in any of the strains of F. necrophorum subsp. funduliforme. In addition, the strains of F. necrophorum subsp. necrophorum were generally more hemolytic than those of F. necrophorum subsp. funduliforme. Lecithinase, beta-lactamase, elastase, hyaluronidase, chondroitin sulfatase, and coagulase were not detected in any of the strains. DNase may be used to differentiate between the two subspecies.