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PubMed2000

Sequence analysis of bacterial DNA in the colon and stomach of the Tyrolean Iceman.

The male human body found in an Alpine glacier on September 19, 1991 ("Tyrolean Iceman") has, for the first time in history, given scientists a chance to perform detailed anatomical, histological, and molecular investigations on the organs of a person from the Neolithic Age (5350-5100 B.P.). In the present study, tissue samples aseptically taken from the stomach and the colon of the mummy were utilized for DNA extraction, and the DNA was PCR-amplified, using primer pairs designed to bind to fragments of the 16s ribosomal RNA gene (16s rDNA) of a broad range of bacteria. The PCR products were cloned in plasmid vectors, and the recombinant clones (amplicons) were sequenced. The sequence data were finally used for scanning data libraries containing the corresponding sequences of present-day bacteria, to infer the putative ecophysiology of the ancient ones. The same procedure was repeated on some fragments of grass from the clothing found near the corpse. These fragments were taken as a control of the microbiological situation of the glacier. The results show that the flora of the Iceman's stomach is entirely composed of Burkholderia pickettii, an organism commonly found in aquatic habitats. The colon, on the other hand, contains several members of the fecal flora of humans, such as Clostridium perfringens, C. ghonii, C. sordellii, Eubacterium tenue, and Bacteroides sp. The Iceman's colon, however, was found to contain, rather unexpectedly, also some members of the genus Vibrio. The results are discussed in light of what is known about the preservation of microbial DNA at the Iceman's site and of previous parasitological studies performed on the Iceman himself and on human coprolites.

Animals↗
PubMed1998

Coronavirus infection and demyelination. Sequence conservation of the S-gene during persistent infection of Lewis-rats.

Coronaviruses display a large phenotypic variability, which may be an important factor for diversification and selection. Previous studies have demonstrated that the S-protein is an essential determinant of virulence and pathogenicity. Therefore we studied the S-gene as an indicator molecule for selection processes employing two different MHV-JHM variants. First, Lewis-rats were infected with MHV-JHM-Pi, a variant that causes demyelinating disease after several weeks p.i. It was not possible to isolate infectious MHV-JHM-Pi from such rats, although viral proteins were expressed. The S-gene was rescued directly from brain tissue employing RT-PCR technology. The amplicons were sequenced in bulk or at the level of single clones. We detected no evidence for an increase of S-gene mutants during the length of time. Only few mutations were found at the clonal level. The changes were distributed throughout the analysed S-gene fragments without a predilection in their location. The frequency of mutation remained low within a range of 0.03 to 0.5 mutations per thousand nucleotides. As a second approach, we sequenced the S-genes of viruses isolated from brain tissue infected with MHV-JHM-ts43. Infection of adult Lewis rats with that mutant resulted several weeks to months p.i. in demyelinating encephalomyelitis. The S-gene of this virus contains an insertion of 423 bp in the S1 region, which is identical to a polymorphic region described for MHV-4. In contrast to JHM-Pi, infectious MHV-JHM-ts43 was readily to isolate from brain tissue. The S-gene sequences of virus isolated 45-106 days p.i. from diseased rats were identical with that of the input virus. These results show, that during a persistent infection of Lewis-rats the S-gene was highly conserved.

Animals↗
PubMed2004

Quantitative PCR deconstruction of discrepancies between results reported by different hybridization platforms.

Differences in hybridization platforms used in gene array analysis experiments can lead to significant differences in hybridization results. In this study we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to investigate discrepant results between the National Institute of Environmental Health Sciences cDNA and Affymetrix oligo platforms used to evaluate hepatic gene expression changes in rats exposed to methapyrilene. Caldesmon cDNA platform hybridization results showed decreases in gene expression levels for the high-dose methapyrilene 7-day pooled samples compared with their controls. By contrast, the Affymetrix oligonucleotide platform showed increases in expression levels for these samples. Quantitative gene expression measurements provide an explanation for the discrepancies observed for these samples. In the case of caldesmon, there is a 74-base sequence in the cDNA clone that is absent in the Affymetrix sequence. The amplicon based on the cDNA clone shows > 100-fold suppression relative to the day 7 high-dose methapyrilene-pooled control. These data demonstrate the importance of using a "gold standard," such as qRT-PCR to confirm key hybridization results as well as to understand the sources of discrepancies resulting from different hybridization platforms.

Animals↗
PubMed2004

Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

Acholeplasma↗
PubMed2002

Beta-lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals.

TEM-, SHV-, and OXA-type beta-lactamases were studied by PCR with 124 ampicillin-resistant (AMP(r)) Escherichia coli isolates recovered from foods of animal origin (n = 20) and feces of humans (n = 49) and healthy animals (n = 55). PCR showed that 103 isolates were positive for TEM and negative for SHV and OXA. Three E. coli isolates showed a positive reaction for OXA, and one showed a positive reaction for SHV. The remaining 17 E. coli isolates were negative for the three enzymes by PCR. Fifty-seven of the 103 bla(TEM) amplicons were sequenced. Different molecular variants of bla(TEM-1) were found in 52 isolates: bla(TEM-1a) (n = 9), bla(TEM-1b) (n = 36), bla(TEM-1c) (n = 6), and bla(TEM-1f) (n = 1). Four inhibitor-resistant TEM (IRT) beta-lactamase-encoding genes were also detected: bla(TEM-30c) (IRT-2), bla(TEM-34b) (IRT-6), bla(TEM-40b) (IRT-11), and bla(TEM-51a) (IRT-15). A new bla(TEM) gene, named bla(TEM-95b), which showed a mutation in amino acid 145 (P-->A) was detected. It was found in a food isolate of chicken origin (AMP(r), amoxicillin-clavulanic acid susceptible). The promoter region in 24 bla(TEM) amplicons was analyzed, and the weak P3 promoter was found in 23 of them (bla(TEM-1) in 20 amplicons and bla(TEM-51a), bla(TEM-30c), and bla(TEM-95b) in 1 amplicon each). The strong Pa/Pb promoter was found only in the bla(TEM-34b) gene. No extended-spectrum beta-lactamases were detected. Mutations at position -42 or -32 in the ampC gene promoter were demonstrated in 4 of 10 E. coli isolates for which the cefoxitin MIC was >/=16 micro g/ml. Different variants of bla(TEM-1) and IRT bla(TEM) genes were found among the AMP(r) E. coli isolates from foods and the feces of humans and healthy animals, and a new gene, bla(TEM-95b) (P3), was detected.

Ampicillin↗
PubMed1999

A PCR based method for the identification of equine influenza virus from clinical samples.

In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct.

Animals↗
PubMed2001

Fine-needle aspiration biopsy diagnosis of gastrointestinal stromal tumors using morphology, immunocytochemistry, and mutational analysis of c-kit.

BACKGROUND: Differentiating gastrointestinal stromal tumors (GISTs) from other intramural mesenchymal tumors of the GI tract on fine-needle aspiration biopsies (FNABs) is difficult. Recent studies have shown that GISTs are immunophenotypically and genetically distinct. GISTs exhibit consistent immunohistochemical expression of CD-117 (KIT) and often express activating mutations of this protooncogene. The aim of the current study was to employ immunocytochemistry and mutational analysis of the c-kit gene to aid in the diagnosis of GISTs on FNAB. METHODS: Five endoscopic ultrasound-guided FNABs of gastrointestinal spindle cell neoplasms performed at the Veterans Affairs Medical Center (VAMC) in Portland, Oregon, from 1998-1999 were reviewed. A panel of immunocytochemical stains was performed on each cellblock including CD-117 (KIT), smooth muscle actin (SMA), desmin, S-100, and CD34. Genomic DNA (gDNA) was extracted, and amplification of exons 9, 11, 13 and 17 of c-kit was performed by polymerase chain reaction (PCR) on CD-117 (KIT) and CD34 positive cases. Direct sequencing of amplicons identified the mutations. RESULTS: Five patients were diagnosed with GISTs based on morphology and immunocytochemical positivity for CD-117 and CD34. PCR analysis of c-kit exon 11 revealed three cases with novel-sized PCR bands in addition to the expected wild-type-sized PCR product. Amplicons from these cases contained an in-frame deletion mutation. One of the two cases with wild-type-;sized exon 11 amplicons was found to be heterozygous for a point mutation producing an amino acid substitution (W557R). No mutations in exon 9, 11, 13, or 17 of c-kit were found in the remaining case. CONCLUSIONS: Ancillary techniques such as immunocytochemistry and c-kit gene mutational analysis may aid in the diagnosis of GISTs on FNABs.

Aged↗
PubMed1999

Human herpesvirus 8 infection in patients with POEMS syndrome-associated multicentric Castleman's disease.

The polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes (POEMS) syndrome is a rare multisystemic disorder associated with osteosclerotic myeloma and multicentric Castleman's disease (MCD). Human herpesvirus type 8 (HHV-8) DNA sequences have been detected in lymph nodes of about 40% of human immunodeficiency virus (HIV)-negative patients with MCD, and in bone marrow stromal cells of patients with multiple myeloma. Considering these data, we investigated the presence of HHV-8 in 18 patients with POEMS syndrome (9 with MCD), by nested polymerase chain reaction (N-PCR) to detect DNA sequenses in various cells and tissues obtained by biopsy or at autopsy (13 patients, of whom 7 had MCD), and by an immunofluorescence assay to detect anti-HHV-8 IgG antibodies in blood (18 patients, of whom 9 had MCD). Detection of HHV-8 DNA was performed using three different N-PCR, targeting nonoverlapping regions in open reading frame (ORF) 25 and ORF26. Seven of 13 (54%) POEMS patients had HHV-8 DNA sequences in their tissues, as assessed by all three N-PCR, and 9 of 18 (50%) had circulating anti-HHV-8 antibodies. HHV-8 was mainly detected in the subset of POEMS patients with MCD (6 of 7 [85%] for DNA sequences; 7 of 9 [78%] for antibodies). The percentage of positive N-PCR was higher in lymph nodes than in bone marrow samples (P <.02). Sequencing of amplicons showed a homogeneous restricted variability in the ORF26 region, characteristic of the minority subgroup B defined by Zong, and responsible for isoleucine and glycine substitutions at amino acid positions 134 and 167. These findings strongly suggest an association of HHV-8 infection with POEMS syndrome-associated MCD.

Adult↗
PubMed2003

[D-loop sequence variation of mitochondrial DNA in captive Chinese alligator].

Chinese alligator, Alligator sinensis, is a critically endangered endemic species under legislative protection in China. Result of recent investigations revealed that number of the alligators was continuously declining in the past 50 years and less than 150 individuals were surviving in the wild until 2000 years. In order to prevent the extinguishing of this species, two breeding farms were set up in early 1980s at Xuanzhou county, Anhui Province and Changxing County, Zhejiang Province respectively. After twenty years of breeding efforts, the number of captive individuals has been brought up to more than 9,000 in total, forming two separate captive subpopulations, Xuanzhou subpopulation (XZSP) and Changxing subpopulation (CXSP). Because of lack of the information regarding genetic diversity of the captive populations, 42 captive individuals including 33 individuals from XZSP and 9 from CXSP were sampled randomly to investigate their genetic status for the strategy in the next protection action. PCR method was adopted for amplification of mitochodrial DNA control region using primers designed in this research. After purification of PCR products, all of amplicons were sequenced directly with ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI 310 genetic analyzer. Consequently, 5' end of control region with length of 462 base pair was obtained from 39 samples. Sequence alignment shows there is no any variation site in this range of control region among the individuals assayed here, namely only one haplotype of the region shared by these alligators. This result strongly indicates that the population of captive Chinese alligator is in very poor genetic diversity status. Reasons for the losing of genetic diversity in the population are mainly attributed to population depression and number of individual decreasing sharply in the past 50 years. Another factor accounting for the phenomena is the limit of founder number of captive population. Finally, authors proposed three pieces of advice for the genetic conservation of Chinese alligator.

Alligators and Crocodiles↗
PubMed2003

Direct sequencing and PCR mapping of integrons reveals multiple class 1 integrons in the multiresistant strain Enterobacter cloacae SCH88040794.

We have characterized the variable region of three integrons found in the multiresistant strain Enterobacter cloacae SCH88040794, using polymerase chain reaction and direct sequencing of amplicons. The first integron has the gene cassette arrangement dfrAI-aadA1 in its variable region and the second aadB-oxa9. The third integron is In40 (aacA4-qacF-cmlB-oxa9), which has recently been described. The multiresistance expressed by this strain to amikacin, gentamicin, trimethoprim, sulfonamides, oxacillin, chloramphenicol and quaternary ammonium compounds is due, at least in part, to these three integrons.

Anti-Bacterial Agents↗
PubMed1997

Molecular detection of an importation of type 3 wild poliovirus into Canada from The Netherlands in 1993.

During the fall and winter of 1992-1993 an outbreak of wild poliovirus type 3-associated poliomyelitis involving 71 patients occurred in The Netherlands. Almost all of the individuals involved in the outbreak belonged to an orthodox religious denomination that prohibits vaccination. A surveillance was initiated to determine if there had been an importation of this same strain of wild poliovirus into a southern Alberta community with a similar religious affiliation. Viral culture of stool samples from consenting individuals in the community resulted in viral isolates which typed as poliovirus type 3. Sequencing of amplicons generated from both the 5' nontranslated region and the VP1/2A portion of the genomes from representative poliovirus isolates indicated a greater than 99% genetic similarity to the strain from The Netherlands. The results of this study show that the utilization of PCR-based diagnostics offers an important molecular tool for the concise and rapid surveillance of possible cases of wild poliovirus importation into communities with individuals at risk for infection.

Adolescent↗
PubMed2026

Ecological and methodological insights from genetic and coprological profiling of gastrointestinal communities in wild howler monkeys.

The gastrointestinal tract hosts a complex community of microorganisms and helminth parasites that collectively contribute to host health and fitness. Analysis of these communities provides insight into diverse aspects of host dietary ecology, immunity, nutrition, and host-parasite interactions. However, research methodologies, such as sample preservation and sequencing approach, can influence how we understand and characterize these features. Here, we profiled the gastrointestinal microbial and helminth communities in different groups of wild Costa Rican mantled howler monkeys (Alouatta palliata palliata). We compared samples stored in ethanol versus directly flash frozen, and contrasted conclusions drawn from 16S versus shotgun sequencing approaches. Bacterial, archaeal, and eukaryotic taxa associated with the digestion of plant material dominated the GI communities. Storage and sequencing methods influenced microbial profiles: ethanol-stored samples exhibited higher diversity than frozen samples, and 16S sequencing detected lower diversity than shotgun. Helminths were detected via coprological microscopy in 71% of individuals, whereas metagenomic detection was inconsistent. This study provides new data on the microorganisms and their putative digestive functions in the gut of a folivorous primate, and highlights the pros and cons of different methodological choices when profiling host-microbiome and host-parasite interactions.

Animals↗
PubMed2006

Detection of mycobacteria in Crohn's disease by a broad spectrum polymerase chain reaction.

BACKGROUND: The role of mycobacterial infection, particularly related to Mycobacterium avium subsp paratuberculosis (Map), in Crohn's disease has long been debated. We developed primer pairs capable of detecting a broad spectrum of mycobacterium and employed them to investigate surgical specimens from patients with Crohn's disease. METHODS: Pan mycobacterium primers of the 65-kDa heat shock protein gene (Hsp65) were used in a polymerase chain reaction (PCR) to examine 12 surgically-resected, formalin-fixed, paraffin-embedded specimens from 11 patients with Crohn's disease. The DNA sequences of amplicons were aligned with those in GenBank. RESULTS: Mycobacterial DNA was found in specimens from three of 11 patients. M. mucogenicum was identified in a specimen from one patient and M. tuberculosis in two, but Map was not identified in any. CONCLUSION: Hsp65-based PCR can be employed to search for occult mycobacterial infection of the gastrointestinal tract in patients with a diagnosis or suspicion of Crohn's disease. This approach may have a therapeutic implication.

Adult↗
PubMed1995

Molecular epidemiology of an outbreak of hepatitis A in Italy.

The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived from the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB.

Antigens, Viral↗
PubMed2002

Resistance to macrolides in clinical isolates of Streptococcus pyogenes due to ribosomal mutations.

OBJECTIVE: Two clinical strains of Streptococcus pyogenes, 237 and 544, one isolated in Slovakia and the other in Croatia, that were resistant to azithromycin (MIC 8 and 2 mg/L, respectively) but susceptible to erythromycin (MIC 0.5 and 0.12 mg/L, respectively) did not contain any gene known to confer macrolide resistance by ribosomal modification (erm gene) or efflux [mef(A) and msr(A) genes]. The aim of the study was to determine the mechanisms of macrolide resistance in both strains. METHODS: Portions of genes encoding ribosomal proteins L22 and L4, and 23S rRNA (domains II and V) in the two macrolide-resistant strains and in control strains susceptible to macrolides, were analysed by PCR and single-strand conformational polymorphism, to screen for mutations. The DNA sequences of amplicons from resistant strains that differed from those of susceptible strains, in terms of their electrophoretic migration profiles, were determined. RESULTS: S. pyogenes 237 displayed a KG insertion after position 69 in ribosomal protein L4. S. pyogenes 544 contained a C2611U mutation in domain V of 23S rRNA. CONCLUSION: Mutations at a similar position in ribosomal protein L4 and 23S rRNA have been reported previously in macrolide-resistant pneumococci. This report shows that similar mutations can be found in macrolide-resistant S. pyogenes.

Adult↗
PubMed2003

[Molecular-genetic comparison of morbilliviruses which caused epizooty in Baikal (Phoca siberica) and Caspian (Phoca caspica) seals].

The nucleotide sequences were determined for a phosphoprotein gene fragment of canine distemper virus (CDV) by using the RT-PCR method with the subsequent sequencing of amplicons from total RNA isolated from 2 samples of Caspian seals, 15 samples of Baikal seals and from samples of dog's and sea-lion's brains. The above materials were phylogenetically analyzed. The heterogeneity of the virus circulating in the Baikal-seal population was demonstrated. Morbillivirus, that caused epizooty in Caspian seals, was shown to be a CDV variant, whose phosphoprotein gene structure was not different, within the analyzed stretch, from the corresponding gene of the most widespread variant of the Baikal seal virus. The data obtained suggest that morbillivirus could be transmitted by birds during their seasonal migrations.

Animal Diseases↗
PubMed2003

Development of a PCR assay for the detection of animal tissues in ruminant feeds.

The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.

Animal Feed↗
PubMed1995

Molecular comparison of coxsackie A virus serotypes.

Genetic diversity of coxsackie A viruses (CAVs) and enteroviruses 68, 69, and 71 was studied by comparing nucleotide and derived amino acid sequences from the 5' untranslated region (5'UTR), VP4-VP2 capsid protein and polymerase 3D region, and 3'UTR. The data were obtained by sequencing PCR amplicons. According to the molecular analysis of the coding region, CAVs belong to three different genetic clusters. CAV2, 3, 5, 7, 8, 10, 12, 14, and 16 form a coherent genetic group designated cluster A. No other enteroviruses, apart from enterovirus 71, are found in this cluster. CAV9 is the only member of the subgroup found in the same genetic group with coxsackie B viruses, the major group of echoviruses and enterovirus 69 (cluster B). Cluster C includes CAV1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 which are genetically close relatives of polioviruses. Enterovirus 68 is related to enterovirus 70 (cluster D). Clusters different from those in the coding region are found in the 5'UTR while the grouping in the 3'UTR is similar to that found in the capsid region. Correlation of these findings with evolution, pathogenesis, and classification is discussed.

Base Sequence↗