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PubMed2003

[Porcine malignant catarrhal fever: diagnostic findings and first detection of the pathogenic agent in diseased swine in Switzerland].

For the first time Ovine Herpesvirus 2 (OvHV-2) was identified in Swiss pigs as the causative agent of Porcine Malignant Catarrhal Fever (MCF). Diseased animals from two farms were observed to show weakness, anorexia, fever up to 41 degrees C, and neurological symptoms, i.e. ataxia, convulsions and hyperesthesia, erosion on the snout and in the oral and nasal mucosa, as well as multiple skin lesions. Histopathological findings included severe non-purulent inflammation with mononuclear cell infiltration in several organs. Most dominant were meningo-encephalitis, disseminated nephritis as well as purulent catarrhalic bronchopneumonia. The findings were quite reminiscent of the lesions due to MCF in cattle and give therefore substantial proof to use Porcine Malignant Catarrhal Fever as the term for the disease. Identification of the causative agent was done with a quantitative PCR specific for OvHV-2. Different tissues from diseased animals were positive. Furthermore, one animal which had been ill for more than five days tested positive for antibodies against an epitope conserved among MCF viruses. Serum samples from diseased animals reacted negative towards Classical Swine Fever- and Pseudorabies virus antigen. A weakly positive reaction against porcine enterovirus type I argued against the involvement of enteroviruses in the observed disease. Moreover, by means of different conventional PCRs, we detected the newly discovered porcine lymphotropic herpesviruses for the first time in Switzerland and could at the same time exclude their involvement in Porcine Malignant Catarrhal Fever.

Animals↗
PubMed2006

Circadian oscillation of innate immunity components in mouse small intestine.

The digestive system is a major port of entry for pathogens. To detect and combat pathogens, the innate immunity in the gut utilizes pattern recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) proteins, and broad-spectrum anti-bacterial polypeptides, such as defensins. We have previously shown that mouse enteric defensins (cryptdins) oscillate around the circadian cycle and peak at the end of the dark phase suggesting control by the biological clock. As the core mechanism of the biological clock has never been studied in the small intestine, our objective was to determine whether the biological clock is functional in mouse jejunum and examine whether mTlr and mNod2 mRNAs, similarly to cryptdins, oscillate throughout the circadian cycle. Mouse jejunum and Paneth-enriched crypt base cells were isolated around the circadian day and the levels of clock (mClock, mBmal1, mPer1, mPer2, mCry1) and innate immunity component (mTlr2, mTlr3, mTlr4, mTlr5, mTlr9, mNod2) genes were measured by real-time PCR. Analysis of mouse jejunum and Paneth-enriched crypt base cells revealed that all clock genes exhibited circadian oscillation. Similarly to cryptdins, mTlr2, mTlr3, mTlr4, mTlr5 displayed circadian rhythmicity in mouse jejunum. Although no circadian oscillation could be detected for mTlr9 and mNod2 in the whole jejunum, these genes oscillated in Paneth-enriched crypt base cells. In addition, mTlr3 exhibited the highest expression level. As the clock regulates intestinal motility and function, resetting of the clock in the small intestine may help not only to restore activity but also to gain better protection against pathogens.

Animals↗
PubMed1991

[Molecular diagnosis in respiratory infections].

Recent advance in molecular biology has enabled the specific and rapid diagnosis of the various infectious diseases. Though we commonly use the three major diagnostic procedure as isolation of the pathogen, direct detection of the pathogen and measurement of the immunological host reaction, DNA probe method would be the fourth major procedure in the clinical microbiology. The indication of the DNA probe method would be considered in the four cases as follows, 1. necessity of the special equipment to isolate the pathogen, 2. necessity of the long period to isolate the pathogen, 3. existence of the cross reaction among the pathogen and relative organisms in the immunological procedure, 4. existence of the difficulty to identify the species of the pathogen by the ordinary procedure. When we consider those indications, Legionnaires' disease might be one of the typical infectious disease to show the benefits of the DNA probe method in diagnosis. So far two types of DNA probe kits for Legionnaires' disease are available. One is the genus specific direct detection kit from the clinical specimens (Gen-probe), and the other is the microplate hybridization kit to identify each species of Legionella. The results of the evaluations of both kits showed the high specificity, rapidity and the clinical usefulness. In the next few years, various types of DNA probe kits might be newly developed and the contribution of those in the clinical microbiology would be much more than we expected.

Aged↗
PubMed2002

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum.

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.

Animals↗
PubMed2006

Prospective surveillance for atypical pathogens in children with community-acquired pneumonia in Japan.

A total of 141 children with community-acquired pneumonia (CAP) were studied prospectively to determine the causative microorganisms. Microbial investigations included examination of postnasal swabs, cultures, polymerase chain reaction (PCR), and serology. The atypical pathogens occurring most frequently were Mycoplasma pneumoniae (58 patients [41.1%]), Chlamydia pneumoniae (4 patients [2.8%]), and concurrent occurrence of both pathogens (1 patient [0.7%]). Patients aged under 4 years showed a relatively lower rate of atypical bacterial etiology compared with those aged 4 years or older. Major bacterial pathogens were detected in 89 patients (atypical pathogens were detected in 28 patients simultaneously), including Streptococcus pneumoniae in 34 patients, Haemophilus influenzae in 60, Moraxella catarrhalis in 48, and multiple pathogens in 42. In patients suspected of having atypical pneumonia, macrolides are recommended.

Adolescent↗
PubMed1996

A modified PCR-based method for rapid non-radioactive detection of clinically important pathogens.

We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.

Bacteria↗
PubMed2003

Pathogenic archaea: do they exist?

Archaea are microorganisms that are distinct from bacteria and eukaryotes. They are prevalent in extreme environments, and yet found in most ecosystems. They are a natural component of the microbiota of most, if not all, humans and other animals. Despite their ubiquity and close association with humans, animals and plants, no pathogenic archaea have been identified. Because no archaeal pathogens have yet been identified, there is a general assumption that archaeal pathogens do not exist. This review examines whether this is a good assumption by investigating the potential for archaea to be or become pathogens. This is achieved by addressing: the diversity of archaea versus known pathogens, opportunities for archaea to demonstrate pathogenicity and be detected as pathogens, reports linking archaea with disease, and immune responses to archaea. In addition, molecular and genomic data are examined for the presence of systems utilised in pathogenesis. The view of this report is that, although archaea can presently be described as non-pathogenic, they have the potential to be (discovered as) pathogens. The present optimistic view that there are no archaeal pathogens is tainted by a severe lack of relevant knowledge, which may have important consequences in the future.

Animals↗
PubMed2002

[Detection of periodontal pathogenic microorganisms in atheromatous plaque. Preliminary results].

INTRODUCTION: Recent studies suggest that chronic infections, including those associated with periodontitis, increase the risk for coronary vascular disease. We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis. MATERIALS AND METHODS: To test this hypothesis, 34 human specimens obtained during carotid endarterectomy or bypass procedures were examined by use of specific oligonucleotide primers for Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Bacteroides forsythus in polymerase chain reaction (PCR) assays. RESULTS: Twenty (59%) of the 34 specimens tested positive for bacterial 16S rDNA. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 32.4% of the 34 atheromas tested positive for at least one of the target periodontal pathogens. Further analysis of the results in the bacterial positive group (n = 20) shows that 55% of the atheromas tested positive for at least one of the target periodontal pathogens. CONCLUSION: These findings indicate that periodontal pathogens are present in atherosclerotic plaques, where they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.

Adult↗
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↗
PubMed2003

Detection of bloodstream pathogens in a bacille Calmette-Guérin (BCG)-vaccinated pediatric population in Malawi: a pilot study.

Children in Malawi receive bacille Calmette-Guérin (BCG) vaccination within the first 3 days of life. Thus, we hypothesized that Malawian children infected with the human immunodeficiency type 1 virus (HIV-1) might be particularly vulnerable to dissemination of the BCG Mycobacterium bovis strain with which they were vaccinated. Following informed consent by parents, we studied children admitted to a Malawi general hospital during the 1998 wet and dry seasons. Blood from cohorts of acutely ill children was cultured for bacteria, including mycobacteria, and fungi, and tested for anti-HIV-1 antibodies. It was shown that non-typhi Salmonella and Escherichia coli were the predominant bloodstream pathogens during the wet and dry seasons, and that bloodstream dissemination of the BCG M. bovis strain is uncommon in HIV-1-infected children who receive the BCG vaccine.

AIDS-Related Opportunistic Infections↗
PubMed2005

Mutagenesis of all eight avr genes in Xanthomonas campestris pv. campestris had no detected effect on pathogenicity, but one avr gene affected race specificity.

Suppression subtractive hybridization (SSH) was used to identify genes present in the systemic crucifer black rot pathogen Xanthomonas campestris pv. campestris 528T but missing from the nonsystemic crucifer leaf spot pathogen, X. campestris pv. armoraciae 417. Among the DNA fragments unique to 528T was Xcc2109, one of eight putative avr genes identified in the published 528T genome (NC_003902). Individual and sequential deletion, insertion mutations, or both of all eight 528T avr gene loci were made, but no change in pathogenicity was observed with any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants affecting the Xcc2109 locus lost avirulence (i.e., became virulent) on Florida Mustard, an X. campestris pv. campestris race-determining, differential host. The Xcc2109 open reading frame as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was cloned and found to complement the Xcc2109 mutants and to confer avirulence to two additional wild-type X. campestris pv. campestris strains, thereby changing their races. Resistance in Florida Mustard to 528T strains carrying avrXccFM occurred without a typical hypersensitive response (HR) on leaves, although a vascular HR was observed in seedlings.

Bacterial Proteins↗
PubMed1993

A PCR primer-specific to Cylindrocarpon heteronema for detection of the pathogen in apple wood.

An oligonucleotide primer (ChInt) was synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Cylindrocarpon heteronema. PCR with primers ChInt and ITS4 (from a conserved sequence of the rDNA) amplified a 470-bp fragment from several isolates of C. heteronema but not from various apple wood saprophytes. Amplification of this fragment was achieved from 1-2 pg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from cankered wood but only after impurities were removed from the DNA on a Qiagen tip-5 column. Southern hybridization analysis confirmed the 470-bp fragment from C. heteronema DNA and cankered wood to be identical.

Base Sequence↗