The use and implications of ribosomal DNA sequencing for the discrimination of digenean species

In just over a decade, the use of molecular approaches for the recognition of parasites has become commonplace. For trematodes, the internal transcribed spacer region of ribosomal DNA (ITS rDNA) has become the default region of choice. Here, we review the findings of 63 studies that report ITS rDNA sequence data for about 155 digenean species from 19 families, and then review the levels of variation that have been reported and how the variation has been interpreted. Overall, complete ITS sequences (or ITS1 or ITS2 regions alone) usually distinguish trematode species clearly, including combinations for which morphology gives ambiguous results. Closely related species may have few base differences and in at least one convincing case the ITS2 sequences of two good species are identical. In some cases, the ITS1 region gives greater resolution than the ITS2 because of the presence of variable repeat units that are generally lacking in the ITS2. Intraspecific variation is usually low and frequently apparently absent. Information on geographical variation of digeneans is limited but at least some of the reported variation probably reflects the presence of multiple species. Despite the accepted dogma that concerted evolution makes the individual representative of the entire species, a significant number of studies have reported at least some intraspecific variation. The significance of such variation is difficult to assess a posteriori, but it seems likely that identification and sequencing errors account for some of it and failure to recognise separate species may also be significant. Some reported variation clearly requires further analysis. The use of a yardstick to determine when separate species should be recognised is flawed. Instead, we argue that consistent genetic differences that are associated with consistent morphological or biological traits should be considered the marker for separate species. We propose a generalised approach to the use of rDNA to distinguish trematode species.

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.

Expression of prostate-specific antigen variant MRNA transcripts in prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA) is important in tumour detection, monitoring disease progression and tumour recurrence. however, PSA is not a cancerspecific marker as levels can also be elevated in benign prostatic disease. A number of different mRNA transcripts of PSA have also been identified in prostatic tissue, but have not been fully characterized (PSA 424, PSA 525, Schulz transcript). Tissue specimens from transurethral resection of the prostate (TURP) or radical prostatectomy were obtained from 17 men with BPH and 15 men with prostate cancer. Total RNA was extracted, and reverse-transcriptionpolymerase chain reaction (RT-PCR) and Southern analysis carried out using transcript-specific primers and probes to determine which mRNA PSA transcripts were expressed. Real-time PCR was performed to determine transcript levels between the two groups using transcript-specific primers and SYBR green fluorescence. Values obtained were normalized to a standard housekeeping gene, B2-microglobulin. Transcripts amplified by RT-PCR and real-time PCR were confirmed by DNA sequencing. Our results show that the transcripts were present in some, but not all, BPH and cancer samples indicating that they are not specific to either BPH or cancer. Analysis of real-time PCR normalized values using a Student’s t -test, shows that there is a significant difference between the two groups for PSA 424, but not wild-type PSA, PSA 525 or the Schulz transcript. Although a larger cohort of samples is needed to further confirm these results, these findings suggest that mRNA levels of PSA 424 may have some utility as a diagnostic or prognostic marker in prostate cancer detection.

Speciation of Antechinus stuartii and A. subtropicus (Marsupialia : Dasyuridae) in eastern Australia: molecular and morphological evidence

This paper evaluates the systematic status of the Antechinus populations of northern New South Wales and southern Queensland, using a combined morphological and molecular (allozymes and mitochondrial DNA) approach. Analysis of the d-loop section of the mitochondrial DNA control region revealed two highly supported clades within A. stuartii sensu lato that were sympatric in the Border Ranges of northern New South Wales. However, genetic distances between these clades were small ( approximately 3%), indicating that time of divergence was probably relatively recent. Allozyme electrophoresis also showed very small differences between clades/ species. Analyses of cranial and dental characters showed that the members of each of these clades differed morphologically and that the clades corresponded to A. stuartii and the recently described A. subtropicus. The combined results support the species status of A. stuartii and A. subtropicus, and suggest that speciation was likely a result of a recent vicariant event.

Simultaneous detection and differentiation of human Polyomaviruses JC and BK by a rapid and sensitive PCR-ELAHA assay and a survey of the JCV subtypes within an Australian population

Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol-PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory. (C) 2004 Wiley-Liss, Inc.

Isolation and characterization of integron-containing bacteria without antibiotic selection

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer

Aims: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. Methods and Results: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced L-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. Conclusions: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. Significance and Impact of the Study: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.

The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract

Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the IS isolates that produced L- and/or D-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the majority of the isolates were closely related to species within the genus Lactobacillus, including Lactobacillus salivarius, Lactobacillus mucosae and Lactobacillus delbrueckii. Four isolates were closely related to Mitsuokella jalaludinii. Lactic acid producing bacteria (LAB) from the equine gastrointestinal tract was dominated by representatives from the genus Lactobacillus, but also included D-lactate-producing bacteria closely related to M. jalaludinii. Identification and characterization of LAB from the equine gastrointestinal tract should contribute to our understanding and management of fermentative acidosis, ulceration of the stomach and laminitis. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates Arthrobacter sp 2AC and Comamonas sp 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75-90% of the initial TOC (total organic carbon) was mineralized, 5-20% remained as DOC (dissolved organic carbon) and 3-10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0-19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with either Arthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.

Novel variants in growth differentiation factor 9 in mothers of dizygotic twins

Context: Genes from the ovarian bone morphogenetic signaling pathway (GDF9 and BMP15) are critical for normal human fertility. We previously identified a deletion mutation in GDF9 in sisters with spontaneous dizygotic (DZ) twins, but the prevalence of rare GDF9 variants in twinning families is unknown. Objective: The objective was to evaluate the frequency of rare variants in GDF9 in families with a history of DZ twinning. Design and Subjects: We recruited 3450 individuals from 915 DZ twinning families (1693 mothers of twins) and 1512 controls of Caucasian origin. One mother of DZ twins was selected from 279 of the 915 families, and a DNA sample was screened for rare variants in GDF9 using denaturant HPLC. Variants were confirmed by DNA sequencing and genotyped in the entire sample by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results: We found two novel insertion/deletions (c.392-393insT, c.1268-1269delAA) and four missense alterations in the GDF9 sequence in mothers of twins. Two of the missense variants (c.307C > T, p.Pro103Ser and c.362C > T, p.Thr121Leu) were located in the proregion of GDF9 and two (c.1121C > T, p.Pro374Leu and c.1360C > T, p.Arg454Cys) in the mature protein region. For each variant, the frequencies were higher in cases compared with controls. The proportion of mothers of DZ twins carrying any variant (4.12%) was significantly higher (P < 0.0001) than the proportion of carriers in controls (2.29%). Conclusion: We describe new variants in the GDF9 gene that are significantly more common in mothers of DZ twins than controls, suggesting that rare GDF9 variants contribute to the likelihood of DZ twinning.

Rapid detection of a chromosomally mediated penicillin resistance-associated ponA mutation in Neisseria gonorrhoeae using a real-time PCR assay

The recent emergence of a decreased susceptibility of Neisseria gonorrhoeae strains to penicillin in New Caledonia has lead clinicians to operate a change in the treatment strategy. In addition, this important health issue has emphasized the need for a rapid means of detecting penicillin resistance in N. gonorrhoeae in order to select an effective treatment and limit the spread of resistant strains. In recent years, the use of fluorescence resonance energy transfer on the LightCycler has proven to be a valuable tool for the screening of mutations occurring in the genome of various microorganisms. In this study, we developed a real-time PCR assay coupled with a fluorometric hybridization probes system to detect a penicillin resistance-associated mutation on the N. gonorrhoeae ponA gene. Following an extensive evaluation involving 136 isolates, melting curve analysis correctly evidenced a 5 degrees C T-m shift in all N. gonorrhoeae strains possessing this mutation, as determined by conventional sequencing analysis. Moreover, the mutation profiles obtained with the real-time PCR showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Overall, our molecular assay allowed an accurate and reproducible determination of the susceptibility to penicillin corresponding to a mutation present in all chromosomally mediated resistant strains of N. gonorrhoeae.