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PubMed2006

Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen.

Listeria monocytogenes is an opportunistic intracellular pathogen that has become an important cause of human foodborne infections worldwide. Given its close relationship to other Listeria species and its tendency to produce non-specific clinical symptoms, the availability of rapid, sensitive and specific diagnostic tests for the differentiation of L. monocytogenes from other Listeria species is helpful for selecting appropriate treatment regimens. In addition, with L. monocytogenes comprising a diversity of strains of varying pathogenicity, the ability to precisely track the strains involved in listeriosis outbreaks and speedily determine their pathogenic potential is critical for the control and prevention of further occurrences of this deadly disease. Extensive research in recent decades has revealed significant insights regarding the molecular mechanisms of L. monocytogenes infection. This in turn has facilitated the development of laboratory procedures for enhanced detection and identification of L. monocytogenes, and has also contributed to the implementation of improved control and prevention strategies against listeriosis. The purpose of this review is to summarize recent progress in the species-specific identification, subtyping and virulence determination of L. monocytogenes strains, and to discuss future research needs pertaining to these important areas of listeriosis.

Animals↗
PubMed2025

Avirulence depletion assay: Combining R gene-mediated selection with bulk sequencing for rapid avirulence gene identification in wheat powdery mildew.

Wheat production is threatened by multiple fungal pathogens, such as the wheat powdery mildew fungus (Blumeria graminis f. sp. tritici, Bgt). Wheat resistance breeding frequently relies on the use of resistance (R) genes that encode diverse immune receptors which detect specific avirulence (AVR) effectors and subsequently induce an immune response. While R gene cloning has accelerated recently, AVR identification in many pathogens including Bgt lags behind, preventing pathogen-informed deployment of resistance sources. Here we describe a new "avirulence depletion (AD) assay" for rapid identification of AVR genes in Bgt. This assay relies on the selection of a segregating, haploid F1 progeny population on a resistant host, followed by bulk sequencing, thereby allowing rapid avirulence candidate gene identification with high mapping resolution. In a proof-of-concept experiment we mapped the AVR component of the wheat immune receptor Pm3a to a 25 kb genomic interval in Bgt harboring a single effector, the previously described AvrPm3a2/f2. Subsequently, we applied the AD assay to map the unknown AVR effector recognized by the Pm60 immune receptor. We show that AvrPm60 is encoded by three tandemly arrayed, nearly identical effector genes that trigger an immune response upon co-expression with Pm60 and its alleles Pm60a and Pm60b. We furthermore provide evidence that Pm60 outperforms Pm60a and Pm60b through more efficient recognition of AvrPm60 effectors, suggesting it should be prioritized for wheat breeding. Finally, we show that virulence towards Pm60 is caused by simultaneous deletion of all AvrPm60 gene paralogs and that isolates lacking AvrPm60 are especially prevalent in the US thereby limiting the potential of Pm60 in this region. The AD assay is a powerful new tool for rapid and inexpensive AVR identification in Bgt with the potential to contribute to pathogen-informed breeding decisions for the use of novel R genes and regionally tailored gene deployment.

Triticum↗
PubMed2006

A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens.

A novel fluorescence imaging technique based on deconvolution microscopy and spectral analysis is presented here as an alternative to confocal laser scanning microscopy. It allowed rapid, specific and simultaneous identification of five major opportunistic pathogens, relevant for public health, in suspension and provided quantitative results.

Bacteria↗
PubMed1997

T cells of lupus and molecular targets for immunotherapy.

A major advance in understanding the basic mechanism driving the pathogenic autoimmune response in SLE has been the identification of nucleosome as a primary immunogen. The production of pathogenic antinuclear antibodies in SLE is mediated by a MHC class II restricted, cognate interaction between select populations of autoimmune T helper cells and autoimmune B cells that recognize epitopes in the different molecular components of the nucleosome particle: a form of intermolecular-intrastructural help. In the SNF1 model, we have localized the critical peptide autoepitopes for lupus nephritis-inducing Th cells in the core histones of nucleosomes, at amino acid positions 10-33 of H-2B and 16-39 and 71-94 of H4. Remarkabely, the nephritogenic epitopes are located in the regions of histones that are also targeted by lupus B cells, as well as the sites where the histones contact DNA in the nucleosome, indicating that they are specially protected during antigen processing. Identification of the peptide epitopes is a basic step toward defining how the pathogenic Th cells emerge in lupus. In addition, we found that the pathogenic Th cells and B cells of lupus have a regulatory defect in the expression of CD40 ligand (CD40L or gp39), which results in abnormal costimulatory signals that sustain the production of pathogenic autoantibodies. Specific immunotherapy that blocks the pathogenic T and B cell interaction in lupus can be designed based on the knowledge of these disease mechanisms.

Animals↗
PubMed2006

Identification and characterization of merozoite antigens of a Theileria species highly pathogenic for small ruminants in China.

A new pathogenic Theileria species transmitted by Haemaphysalis qinghaiensis was identified in the Northwestern part of China and was shown to be highly pathogenic for small ruminants. The present article aimed at identifying merozoite antigens that might be suitable for developing diagnostic methods and designing a potential vaccine. Absence of other theilerial or babesial infections was confirmed by reverse line blot in all antigen samples used. Extensive Western blot analyses using serum from infected and noninfected animals led to the identification of four potential merozoite immunoreactive proteins at different molecular weights. Further protein characterization using peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching resulted in two significant hits that identified two proteins of parasite origin, one homologous to a possible MO25-family protein from Cryptosporidium parvum and the other with an HSP70 from Theileria annulata. Another protein was also identified as a parasite protein but without significant homology. Immunization of rabbits with selected proteins produced antisera that reacted specifically on Western blots with merozoite antigens of the corresponding sizes. This article represents the first identification and characterization of potential antigenic proteins of Theileria sp. (China) for veterinary purposes.

Animals↗
PubMedDate not supplied

Identification of Candida albicans genes that induce Saccharomyces cerevisiae cell adhesion and morphogenesis.

Morphogenesis and adhesion to host tissues and medical devices contribute to the virulence of Candida albicans, the most common fungal pathogen isolated from humans. However, identification of molecular mechanisms of C. albicans adhesion and morphogenesis has been impaired by the lack of effective molecular and genetic tools available for this organism. Saccharomyces cerevisiae provides an attractive model system for studying C. albicans adhesion and morphogenesis because of its well-characterized genetics and gene expression systems. To gain insight into the genetic mechanisms of C. albicans adhesion and morphogenesis, we used a parallel plate flow chamber to screen and quantitatively characterize attachment to polystyrene of an adhesion-deficient nonfilamentous flo8Delta S. cerevisiae strain expressing a C. albicans genomic library. We identified six C. albicans genes that are capable of promoting cell adhesion and pseudohyphal development in S. cerevisiae. We also analyzed the ability of these adhesion-promoting genes to regulate the expression of FLO11, which encodes an endogenous S. cerevisiae adhesin. One C. albicans gene, EAP1, appears to directly mediate adhesion and morphogenesis while the remaining five (EAP2, SWI1, MSB1, AAF1, and TEC1) upregulate expression of endogenous S. cerevisiae adhesins. These results suggest that S. cerevisiae is a useful system for molecular characterization of factors that regulate C. albicans adhesion and morphogenesis and that parallel plate flow chamber-based adhesion assays can be used in conjunction with genetic screens to identify molecular mechanisms regulating fungal cell adhesion.

Biotechnology↗
PubMed2006

Incidence of Leptosphaerulina crassiasca in symptomless leaves of peanut in southern India.

A Loculoascomycete fungus belonging to the genus Leptosphaerulina was found to survive as symptomless endophyte in the leaflets and rachis of peanut plant (TMV 7). Based on morphological and colony characteristics, it is identified as Leptosphaerulina crassiasca, a fungus that causes the pepper spot and leaf scorch diseases in peanut. Although several common endophytic fungi were recovered from the leaves of peanut, L . crassiasca showed a high relative density of infection. Owing to its endophytic nature, its pathogenicity could not be proved by infection studies. However, its identification as L. crassiasca suggests that this pathogen survives as symptomless endophyte in its host. There are limited studies on fungal endophytes of cultivated crops and hence, the present observation underlines the need to know more about the biology of endophytic fungi of crop plants.

Arachis↗
PubMed2006

Genomic approaches to understanding bacterial virulence.

The genomic sequences of bacterial pathogens and of the host species they infect have greatly increased the understanding of host-pathogen interactions. Sequences of bacterial genomes have led to the identification of virulence factors through the use of bioinformatics, targeted mutant library construction, screening approaches combining transposon mutagenesis and microarray technology, and through the expression of libraries of bacterial proteins within model organisms such as yeast. Host genomic information has also yielded insights into bacterial virulence through transcriptional profiling of host responses to infection and identification of host proteins required for bacterial pathogenicity using knockdown of host gene product expression during infection. Research using genomic approaches to bacterial pathogenesis is a rapidly growing field and will expand further as additional bacterial genome sequences become available and techniques for conducting high-throughput analysis are refined.

Bacteria↗
PubMed2003

A review of porcine tonsils in immunity and disease.

The porcine tonsils are a group of lymphoepithelial tissues located at the common openings of the gastrointestinal and respiratory tracts. The tonsils participate in a variety of functions involving innate, cellular and humoral immunity at the local and systemic levels. Among these immunological functions is the continuous surveillance for the presence of foreign antigens at the openings of the gastrointestinal and respiratory tracts. Within the pig, the movement of lymphocytes, cytokines and chemotactic molecules from the tonsils to other lymphoid organs confers immunity to other portals of pathogen entry and facilitates an efficient and rapid systemic immune response. In spite of the immunological nature of the tonsils, some microorganisms have acquired adaptations that allow them to circumvent the tonsillar immune defenses and utilize the tonsils as a site of entry, replication and colonization. Several bacterial and viral pathogens persist asymptomatically within the tonsils, making identification of asymptomatic carrier animals difficult in disease control and/or pathogen elimination. This paper reviews the current information on the anatomy, immunology and pathobiology of porcine tonsils and discusses the tonsils as a site of pathogen entry, replication and colonization using Salmonella spp., classical swine fever virus and porcine reproductive and respiratory syndrome virus as examples.

Animals↗
PubMed2006

Identification of differentially expressed genes in Malus domestica after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere.

Biological control of plant diseases by the application of antagonistic micro-organisms to the plant phyllosphere is only marginally understood. Suppression subtractive hybridization (SSH) was used for the identification of genes expressed after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere of the apple scab-susceptible cultivar Malus domestica cv. Holsteiner Cox. In total, 157 expressed sequence tag (EST) clones were obtained. The sequencing of 113 ESTs which have a significantly elevated transcript level and the comparison of the obtained sequences with databases revealed similarities to different classes of pathogenesis-related proteins, for example, RNase-like PR10 protein and endochitinase, or similarities to proteins expressed under stress conditions that could have a protective function, for example, a germin-like protein, glutathione S-transferase, thioredoxin-like proteins, and heat shock proteins. In addition, several transcripts were identified that code for proteins which have a crucial role at different stages of pathogen recognition and in signalling pathways or an as yet unknown function in plant defence. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated after pathogen infection are also up-regulated after the application of a non-pathogenic bacterium to a M. domestica cultivar. The expression of these proteins might increase the plant resistance towards pathogen infection and damage.

Blotting, Northern↗
PubMed2004

Detection of sorbitol-negative and sorbitol-positive Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Salmonella spp. in dairy farm environmental samples.

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administration's Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.

Animal Husbandry↗
PubMed2000

[Xenografts and the probability of viral zoonotic risk: reflections based on the sanitary control of a pig population free from specific pathogenic organisms and risk analysis].

An analysis of the risk factors involved has been made and a classical approach has been adopted, consisting of at least six stages. In the context of risk assessment and identification of potential danger, the pathogenic agents in the pig have been identified and those which are potentially pathogenic to man have been indicated. The risk of spreading, and exposure to certain pathogenic agents (SEPA) varies depending on the nature of the donor organs. The risk factors should be managed by a quality-control approach, which at the sanitary level should consist of controlling all production levels in the pig population, from rearing to slaughtering, and the subsequent removal of vital organs and tissue for xenotransplantation or xenografts. The personnel involved should also undergo rigorous medical examination.

Animal Husbandry↗
PubMed1992

Microbial etiology of periodontal diseases. Where are we? Where are we going?

Many studies indicate a strong association of Actinobacillus actinomycetemcomitans with localized juvenile periodontitis. Species associated with adult periodontitis include Bacteroides forsythus, Porphyromonas gingivalis, Prevotella intermedia, A. actinomycetemcomitans, and Wolinella recta. Capnocytophaga species may be important in pubertal gingivitis. An unnamed spirochete related to Treponema pallidum has been identified in acute necrotizing ulcerative gingivitis lesions. Species isolated from prepubertal periodontitis, peri-implantitis, pericoronitis, and human immunodeficiency virus gingivitis and periodontitis are similar to those isolated from periodontal and gingival infections. Species identification in combination with clinical characteristics facilitates periodontal diagnosis. DNA probes, immunoassays, and benzoyl-arginine naphthylamide reactivity methods can be used to indicate putative pathogens in plaque samples. Microbial identification aids in antibiotic selection and planning a treatment regimen.

Actinomyces↗
PubMed2006

Identification of Staphylococcus hyicus by polymerase chain reaction mediated amplification of species specific sequences of superoxide dismutase A encoding gene sodA.

A species specific PCR test, based on manganese-dependent superoxide dismutase A encoding gene sodA, was developed for the identification of Staphylococcus hyicus, an important bacterial pathogen in pigs. The designed primers allowed a rapid and reliable identification of phenotypically characterized S. hyicus, isolated in Russia, Germany and Denmark. No cross reactivities could be observed investigating staphylococcal reference strains representing 18 different species and subspecies. The use of the described primers might improve a future diagnosis of this bacterial pathogen.

Bacterial Proteins↗
PubMed1999

Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes.

lisRK encodes a two-component regulatory system in the food pathogen Listeria monocytogenes LO28. Following identification of the operon in an acid-tolerant Tn917 mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.

Animals↗
PubMed1991

Identification of clinical isolates of gram-negative nonfermentative bacteria by an automated cellular fatty acid identification system.

An automated cellular fatty acid (CFA) bacterial identification system, Microbial Identification System (MIS; Microbial ID, Newark, Del.), was compared with a conventional system for the identification of 573 strains of gram-negative nonfermentative bacteria. MIS identifications were based exclusively on the CFA composition following 22 to 26 h of growth at 28 degrees C on Trypticase soy agar. MIS identifications were listed with a confidence measurement (similarity index [SI]) on a scale of 0 to 1.0. A value of greater than or equal to 0.5 was considered a good match. The MIS correctly listed as the first choice 478 of 532 (90%) strains contained in the data base. However, only 314 (59%) had SI values of greater than or equal to 0.5. Of the 54 strains in which there was not agreement, 37 belonged to the genera Acinetobacter, Moraxella, or Alcaligenes or were Pseudomonas pickettii. Reproducibility studies suggest that SI variation is most likely a function of a difference in culture age at the time of analysis, which is due to the relatively low temperature and time of incubation. Other discrepancies were attributable to insufficiently characterized library entries or an inability to differentiate chemotaxonomically closely related species. The MIS, as the first automated CFA identification system, is an accurate, efficient, and relatively rapid method for the identification of gram-negative nonfermentative bacteria. The development of a CFA library with the media and incubation conditions routinely used for the isolation of clinical pathogens could further decrease the identification time and provide an increase in accuracy.

Bacteriological Techniques↗
PubMed2003

Enhancement of equine infectious anemia virus virulence by identification and removal of suboptimal nucleotides.

Pathogenicity was reportedly restored to an avirulent molecular clone of equine infectious anemia virus (EIAV) by substitution of 3' sequences from the pathogenic variant strain (EIAV(PV)). However, the incidence of disease in horses/ponies was found to be significantly lower (P = 0.016) with the chimeric clone (EIAV(UK)) than with EIAV(PV). This was attributable to 3' rather than 5' regions of the proviral genome, where EIAV(UK) differs from the consensus EIAV(PV) sequence by having a 68-bp duplication in the 3' LTR and arginine (R(103)) rather than tryptophan (W(103)) at position 103 in the second exon of rev. In EIAV(UK) recipients the duplication was rapidly eliminated and R(103) replaced by W(103) in the viral population. Furthermore, removal of the 3' variant sequences from EIAV(UK) (EIAV(UK3)) resulted in an equivalent (P = 0.013) disease potential in Equus caballus to EIAV(PV). The 68-bp duplication and/or R(103) may limit peak viral RNA accumulation during acute infection.

Amino Acid Sequence↗
PubMed1991

Identification of genes governing filamentous growth and tumor induction by the plant pathogen Ustilago maydis.

Two master regulatory loci, a and b, govern life-cycle transitions of the phytopathogenic fungus Ustilago maydis. Fusion of haploids that differ at both a and b results in production of a filamentous dikaryon, which induces tumors in its host, maize. Here I describe identification of genes distinct from a and b that play roles in these life-cycle transitions. These studies identify three genes, fuz1, fuz2, and rtf1, that are necessary for filament formation. fuz1 is also necessary for normal size and distribution of tumors and for teliospore formation; fuz2 is also necessary for teliospore germination. Mutations in the rtf1 gene, which are recessive, bypass the requirement of different b alleles for tumor formation. This observation indicates that rtf1 codes for a negative regulator of tumor induction. The fuz1, fuz2, and rtf1 genes may be targets for the a and b loci.

Alleles↗