Biomedical subjects

R Wayne Litaker

Publications and source records attributed to R Wayne Litaker.

5 recordsLinked to original sources

PubMed2006

Rapid one-step quantitative reverse transcriptase PCR assay with competitive internal positive control for detection of enteroviruses in environmental samples.

Human enteroviruses can serve as a more accurate indicator of human fecal contamination than conventional bacteriological fecal indicators. We describe here a quantitative reverse transcriptase PCR (qRT-PCR) assay specifically tailored to detect these viruses in environmental waters. The assay included a competitive internal positive control (CIPC) that allowed the inhibition of qRT-PCRs to be quantitatively assessed. Coamplification of the CIPC with enteroviral genetic material did not affect the sensitivity, specificity, or reproducibility of the enteroviral qRT-PCR assay. The assay is rapid (less than 5 h from sample to result), has a wide dynamic range (>3 logs), and is capable of detecting as few as 25 enteroviral genomes with an average amplification efficiency of 0.91. In samples with low or moderate inhibition, the delay in CIPC amplification was used to adjust enterovirus qRT-PCR concentrations to account for losses due to inhibition. Samples exhibiting significant inhibition were not corrected but instead diluted twofold and immediately assayed again. Using significantly inhibited samples, it was found that dilution relieved inhibition in 93% (25 of 27) of the samples. In addition, 15% (4 of 27) of these previously negative samples contained enteroviral genomes. The high-throughput format of the assay compared to conventional culture-based methods offers a fast, reliable, and specific method for detecting enteroviruses in environmental water samples. The ability of the assay to identify false negatives and provide improved quantitative assessments of enterovirus concentrations will facilitate the tracking of human fecal contamination and the assessment of potential public health risk due to enteroviruses in recreational and shellfish harvesting waters.

Base Sequence↗

PubMed2006

Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions.

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.

Animals↗

PubMed2004

Early evolution within kinetoplastids (euglenozoa), and the late emergence of trypanosomatids.

Many important relationships amongst kinetoplastids, including the position of trypanosomatids, remain uncertain, with limited taxon sampling of markers other than small subunit ribosomal RNA (SSUrRNA). We report gene sequences for cytosolic heat shock proteins 90 and/or 70 (HSP90, HSP70) from the potentially early-diverging kinetoplastids Ichthyobodo necator and Rhynchobodo sp., and from bodonid clades '2' (Parabodonidae) and '3' (Eubodonidae). Some of the new cytosolic HSP70 sequences represent a distinct paralog family (HSP70-B), which is related to yet another paralog known from trypanosomatids (HSP70-C). The (HSP70-B, HSP70-C) clade seemingly diverged before the separation between kinetoplastids and diplonemids. Protein phylogenies support the basal placement of Ichthyobodo within kinetoplastids. Unexpectedly, Rhynchobodo usually forms the next most basal group, separated from the clade '1' bodonids with which it has been allied. Bootstrap support is often weak, but the possibility that Rhynchobodo represents a separate early-diverging lineage within core kinetoplastids deserves further testing. Trypanosomatids always fall remote from the root of kinetoplastids, forming a specific relationship with bodonid clades 2 (and 3), generally with strong bootstrap support. These protein trees with improved taxon sampling provide the best evidence to date for a 'late' emergence of trypanosomatids, contradicting recent SSUrRNA-based proposals for a relatively early divergence of this group.

Amino Acid Sequence↗

PubMed2003

Tritrichomonas foetus and not Pentatrichomonas hominis is the etiologic agent of feline trichomonal diarrhea.

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, a n organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

Animals↗

PubMedDate not supplied

Molecular taxonomy of the suborder Bodonina (Order Kinetoplastida), including the important fish parasite, Ichthyobodo necator.

Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male x M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum-likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of L. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.

Animals↗