Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H?/Hnt Isolates from Cattle and Clonal Relationship Analysis

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H−/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx₁ genes combined with the EHEC hlyA (hlyA_EHEC) gene, the eae gene of the ζ subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.Shiga toxin-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (45). In humans, infections with some STEC serotypes may result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by the hemolytic uremic syndrome (HUS) (32). These STEC strains are also designated enterohemorrhagic Escherichia coli (EHEC). Consequently, EHEC strains represent a subgroup of STEC with high pathogenic potential for humans. Although E. coli O157:H7 is the most frequent EHEC serotype implicated in HUS, other serotypes can also cause this complication. Non-O157:H7 EHEC strains including serotypes O26:H11/H−, O103:H2/H−, O111:H8/H10/H−, and O145:H28/H25/H− and sorbitol-fermenting E. coli O157:H− isolates are present in about 50% of stool cultures from German HUS patients (10, 42). However, STEC strains that cause human infection belong to a large number of E. coli serotypes, although a small number of STEC isolates of serogroup O156 were associated with human disease (7). Strains of the serotypes O156:H1/H8/H21/H25 were found in human cases of diarrhea or asymptomatic infections (9, 22, 25, 26). The detection of STEC of serogroup O156 from healthy and diseased ruminants such as cattle, sheep, and goats was reported by several authors (1, 11-13, 21, 39, 46, 50, 52). Additional EHEC-associated virulence genes such as stx, eae, hlyA_EHEC, or nlaA were found preferentially in the serotypes O156:H25 and O156:H− (11-13, 21, 22, 50, 52).Numerous methods exist for the detection of pathogenic E. coli, including genotypic and phenotypic marker assays for the detection of virulence genes and their products (19, 47, 55, 57). All of these methods have the common drawback of screening a relatively small number of determinants simultaneously. A diagnostic DNA microarray based on the ArrayTube format of CLONDIAG GmbH was developed as a viable alternative due to its ability to screen multiple virulence markers simultaneously (2). Further microarray layouts working with the same principle but different gene targets were developed for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria (5) and for the rapid DNA-based serotyping of E. coli (4). In addition, a protein microarray for E. coli O serotyping based on the ArrayTube format was described by Anjum et al. (3).The aim of our study was the molecular genotyping of bovine E. coli field isolates of serogroup O156 based on miniaturized E. coli oligonucleotide arrays in the ArrayStrip format and to combine the screening of E. coli virulence markers, antimicrobial resistance genes, and DNA serotyping targets, some of which were partially described previously for separate arrays (2, 4, 5). The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O156 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.     

Dogs shed Neospora caninum oocysts after ingestion of naturally infected bovine placenta but not after ingestion of colostrum spiked with Neospora caninum tachyzoites

An experiment was carried out to determine whether bovine colostrum or placenta could be a source of infection of Neospora caninum for dogs. For this purpose, two dogs were fed bovine colostrum to which culture-derived N. caninum tachyzoites were added and two other dogs were fed placental cotyledonary tissue from N. caninum seropositive cows. One dog served as a negative control during the start of the experiment but this control dog was fed cotyledonary tissue later on. None of the dogs did produce serum antibodies to N. caninum. All three dogs that were fed cotyledonary tissue did shed N. caninum oocysts, but no oocyst shedding was seen in the two dogs that were fed colostrum with N. caninum tachyzoites. Oocyst excretion did not resume in two dogs after repeated feeding of N. caninum infected placenta. The identity of the oocysts was confirmed by a bioassay in gerbils. It is concluded that ingestion of bovine placenta by dogs is an effective mode of transmission of N. caninum from cattle to dogs.     

Exploratory Hydrocarbon Drilling Impacts to Arctic Lake Ecosystems

Recent attention regarding the impacts of oil and gas development and exploitation has focused on the unintentional release of hydrocarbons into the environment, whilst the potential negative effects of other possible avenues of environmental contamination are less well documented. In the hydrocarbon-rich and ecologically sensitive Mackenzie Delta region (NT, Canada), saline wastes associated with hydrocarbon exploration have typically been disposed of in drilling sumps (i.e., large pits excavated into the permafrost) that were believed to be a permanent containment solution. However, failure of permafrost as a waste containment medium may cause impacts to lakes in this sensitive environment. Here, we examine the effects of degrading drilling sumps on water quality by combining paleolimnological approaches with the analysis of an extensive present-day water chemistry dataset. This dataset includes lakes believed to have been impacted by saline drilling fluids leaching from drilling sumps, lakes with no visible disturbances, and lakes impacted by significant, naturally occurring permafrost thaw in the form of retrogressive thaw slumps. We show that lakes impacted by compromised drilling sumps have significantly elevated lakewater conductivity levels compared to control sites. Chloride levels are particularly elevated in sump-impacted lakes relative to all other lakes included in the survey. Paleolimnological analyses showed that invertebrate assemblages appear to have responded to the leaching of drilling wastes by a discernible increase in a taxon known to be tolerant of elevated conductivity coincident with the timing of sump construction. This suggests construction and abandonment techniques at, or soon after, sump establishment may result in impacts to downstream aquatic ecosystems. With hydrocarbon development in the north predicted to expand in the coming decades, the use of sumps must be examined in light of the threat of accelerated permafrost thaw, and the potential for these industrial wastes to impact sensitive Arctic ecosystems.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Digestibility of Duddingtonia flagrans chlamydospores in ruminants: in vitro and in vivo studies

Background

Rabies is a widespread disease in African domestic dogs and a serious public health problem in developing countries. Canine rabies became established in Africa during the 20th century, coinciding with ecologic changes that favored its emergence in canids. This paper reports the results of a cross-sectional study of dog ecology in the Antananarivo urban community in Madagascar. A questionnaire survey of 1541 households was conducted in Antananarivo from October 2007 to January 2008. The study addressed both owned and unowned dogs. Various aspects of dog ecology were determined, including size of dog population, relationship between dogs and humans, rabies vaccination.

Results

Dog ownership was common, with 79.6 to 94.1% (mean 88.9%) of households in the six arrondissements owning dogs. The mean owned dog to person ratio was 1 dog per 4.5 persons and differed between arrondissements (administrative districts), with ratios of 1:6.0 in the first arrondissement, 1:3.2 persons in the 2^nd, 1:4.8 in the 3^rd, 1:5.2 in the 4^th, 1:5.6 in the 5^th and 1:4.4 in the 6^th arrondissement. Overall, there were more male dogs (61.3%) and the male/female sex ratio was estimated to be 1.52; however, mature females were more likely than males to be unowned (OR: 1.93, CI 95%; 1.39Conclusion Antananarivo has a higher density of dogs than many other urban areas in Africa. The dog population is unrestricted and inadequately vaccinated against rabies. This analysis of the dog population will enable targeted planning of rabies control efforts.     

Seed characteristics and fatty acid composition of castor (<i>Ricinus communis</i> L.) varieties in Northeast China

Oil content and fatty acid composition were investigated on 12 castor varieties and strains by using the soxhlet extraction method and capillary gas chromatography. This was made to provide a reference and theoretical basis for castorbean breeding with high oil content, determine variability of seed compounds for breeding purposes, and broaden chemical material choices. Results revealed that crude fat percentage in seeds ranged from 18.91 to 35.84% with an average of 25.91%; the absolute content of ricinoleic acid varied between 171.65 g/kg and 314.03 g/kg with an average of 222.43 g/kg, and kernel crude fat percentage was between 24.28 and 46.97% with an average of 34.30%. All these study variables were highest in the 2129 strain. The percentage of ricinoleic acid in crude fat was between 83.85 to 87.62%, and the highest value was found in the zhebi4 accession. The other fatty acids appeared in small concentrations, and showed small amplitude: 1.12 to 1.61%, 1.21 to 1.61%, 3.53 to 4.80%, 5.35 to 6.38%, 0.52 to 0.79%, 0.05 to 0.08% and 0.43 to 0.55%, for palmitic, stearic, oleic, linolic, linolenic, arachidic, and arachidonic acids, respectively. One hundred seed weight was determined for each accession. One hundred seed weight ranged from 25.7 g to 34.0 g with an average of 29.9 g. There was a significant correlation between seed weight and oil content, but the correlation value was low (r=0.51). Cluster analysis by SSPS based on the content of fatty acid composition revealed that the accessions were divided into three independent clusters. These findings will clearly provide useful information for further research in breeding and utilization of castor oil.     

Diel movement of smallmouth yellowfish Labeobarbus aeneus in the Vaal River,South Africa

Diel movements of Orange–Vaal smallmouth yellowfish Labeobarbus aeneus (Burchell, 1822) in the Vaal River, South Africa, were determined by externally attaching radio transmitters to 11 adult fish and manually tracking them between March and May 2012. Twenty-four radio telemetry monitoring surveys produced 2 304 diel tracks. At night, yellowfish displayed a preference for slow shallow (<0.3?m s?1, <0.5?m) and fast shallow habitats (>0.3?m s?1, <0.3?m), whereas by day they avoided these habitats, preferring fast deep areas (>0.3?m s?1, >0.3?m). The average total distance of 272?m moved per 24-hour period was three times greater than the diel range, and the average maximum displacement per minute was significantly higher in daytime (4?m) than at night (1.5?m). These findings suggest that L. aeneus is active primarily during the day in fast-flowing, deeper waters, and relatively inactive at night, when it occupies shallower habitats. This behaviour should be further explored to identify causal mechanisms underlying the diel habitat shifts in this species such as water temperature, foraging tactics and/or predator avoidance.     

Membrane TNF confers protection to acute mycobacterial infection

Background

Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).

Methods

C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.

Results

While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.

Conclusion

Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.     

Analysis of the clonal relationship of shiga toxin-producing Escherichia coli serogroup O165:H25 isolated from cattle

Variations in time and space of a clonal group of Escherichia coli O165:H25 on a cattle farm were monitored. The virulence marker pattern (stx genes, eae gene, hly(EHEC) gene, katP gene, espP gene, efa gene) suggests that E. coli O165:H25 of bovine origin may represent a risk for human infection.