A comparative study of cultural methods for the detection of Salmonella in feed and feed ingredients

Background  

Giardia duodenalis is a ubiquitous protozoan parasite that has emerged as a significant opportunistic human pathogen. G. duodenalis may have a deleterious effect on animal growth and performance, therefore its potential as a production limiting organism should not be discounted. We therefore undertook this study to determine management and environmental factors in feedlots that influence the prevalence and environmental load of G. duodenalis cysts in fecal material deposited by feedlot cattle in the central and western United States.     

LOMA: A fast method to generate efficient tagged-random primers despite amplification bias of random PCR on pathogens

Background  

Pathogen detection using DNA microarrays has the potential to become a fast and comprehensive diagnostics tool. However, since pathogen detection chips currently utilize random primers rather than specific primers for the RT-PCR step, bias inherent in random PCR amplification becomes a serious problem that causes large inaccuracies in hybridization signals.     

Analysis of 16S rRNA gene sequences and circulating cell-free DNA from plasma of chronic fatigue syndrome and non-fatigued subjects

Background  

The association of an infectious agent with chronic fatigue syndrome (CFS) has been difficult and is further complicated by the lack of a known lesion or diseased tissue. Cell-free plasma DNA could serve as a sentinel of infection and disease occurring throughout the body. This type of systemic sample coupled with broad-range amplification of bacterial sequences was used to determine whether a bacterial pathogen was associated with CFS. Plasma DNA from 34 CFS and 55 non-fatigued subjects was assessed to determine plasma DNA concentration and the presence of bacterial 16S ribosomal DNA (rDNA) sequences.     

Limited genetic diversity in <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Enteritidis PT13

Background  

Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

Development of amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot

Aims

The objective of this work was to design an amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil.

Methods and Results

Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non‐PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils.

Conclusions

The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen.

Significance and Impact of the Study

These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.     

Adaptation of <Emphasis Type="Italic">Salmonella enterica</Emphasis> Hadar under static magnetic field: effects on outer membrane protein pattern

Background  

Salmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments.     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.     

Generation of a restriction minus enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> E2348/69 strain that is efficiently transformed with large,low copy plasmids

Background  

Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic Escherichia coli strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (~15) bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s), with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen.     

Proteomic analysis of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis following propionate adaptation

Background  

Salmonella Enteritidis is a highly prevalent and persistent foodborne pathogen and is therefore a leading cause of nontyphoidal gastrointestinal disease worldwide. A variety of stresses are endured throughout its infection cycle, including high concentrations of propionate (PA) within food processing systems and within the gut of infected hosts. Prolonged PA exposure experienced in such milieus may have a drastic effect on the proteome of Salmonella Enteritidis subjected to this stress.     

Genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor

Background  

Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia.     

Allele distribution and genetic diversity of VNTR loci in <Emphasis Type="Italic">Salmonella enterica</Emphasis>serotype Enteritidis isolates from different sources

Background  

Salmonella enterica serotype Enteritidis (S. Enteritidis) is a zoonotic pathogen, which can be found in many sources including animals and the environment. However, little is known about the molecular relatedness among S. Enteritidis isolates from different sources. We have applied multiple-locus variable number tandem repeat analysis (MLVA) to study the genetic diversity of S. Enteritidis isolates from human and non-human sources.     

Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR

Aim

Create a method for highly sensitive, selective, rapid and easy‐to‐use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously.

Methods and Results

Test‐systems for real‐time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test‐systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 μl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA^® amplifier.

Conclusions

Preloaded 30‐reaction micromatrices having shelf life of 3 and 6 months (for RNA‐ and DNA‐based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg).

Significance and Impact of the Study

The accurate, rapid and user‐friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.     

Positive selection of HIV host factors and the evolution of lentivirus genes

Background  

Positive selection of host proteins that interact with pathogens can indicate factors relevant for infection and potentially be a measure of pathogen driven evolution.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

The biochemical differentiation of Enterobacter sakazakii genotypes

Background  

Enterobacter sakazakii is an emergent pathogen that has been associated with neonatal infections through contaminated powdered infant milk formula. The species was defined by Farmer et al. (1980) who described 15 biogroups according to the biochemical characterization of 57 strains. This present study compares genotypes (DNA cluster groups based on partial 16S rDNA sequence analysis) with the biochemical traits for 189 E. sakazakii strains.     

Effects of <Emphasis Type="Italic">crp</Emphasis> deletion in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serotype Gallinarum

Background  

Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested.     

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis

Purpose of work  

A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required.     

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.