Wayne G. Woods

Construction and analysis of a recombination‐deficient (radA) mutant of Haloferax volcanii

By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii, we created and characterized a recombination‐deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA/RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination‐deficient background is desired.

Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia

Species of Eimeria from chickens from Australia were characterised using a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) approach. The ribosomal DNA region spanning the second internal transcribed spacer (ITS-2) was amplified from genomic DNA by PCR, digested separately with three restriction endonucleases (CfoI, Sau3AI and TaqI) and the fragments separated by denaturing gel electrophoresis. The PCR products amplified from the six species varied from approximately 70 to 620 bp on agarose gels, with differences in size and number of bands among species, but no apparent variation within a species. The PCR-RFLP analysis of ITS-2 amplicons on denaturing gels gave characteristic profiles for individual species (except for minor variation in profiles within some species). The results indicate that ITS-2 contains useful genetic markers for the identification of six Eimeria species occurring in Australia.

Automated, fluorescence-based approach for the specific diagnosis of chicken coccidiosis

We have established a fluorescence‐based electrophoretic approach for the specific identification of all seven currently recognised species of Eimeriainfecting chickens. The second internal transcribed spacer (ITS‐2) of ribosomal DNA is amplified by polymerase chain reaction (PCR) from any of the seven species using a single set of oligonucleotide primers (of which the reverse one is fluorescently labelled). The amplicons are heat‐denatured, subjected to denaturing polyacrylamide gel electrophoresis in a 377 DNA sequencer (ABI). The chromatograms produced are stored electronically and then analysed using GeneScan 3.1 software. Using control DNA samples representing monospecific lines of Eimeria,regions in the chromatograms have been defined for the specific identification of each of the seven species, although some variation in the chromatograms (reflecting population variation) was detectable within two species. Electrophoretic reading and analysis is carried out automatically using a computer imaging system, thus making it a time‐ and cost‐effective approach. It is well suited for high‐throughput diagnostic screening of oocyst samples and should find applicability as a tool for prevalence studies, monitoring of coccidiosis outbreaks and the quality control of vaccines.

High-resolution electrophoretic procedures for the identification of five Eimeria species from chickens, and detection of population variation

To overcome limitations of conventional approaches for the identification of Eimeria species of chickens, we have established high resolution electrophoretic procedures using genetic markers in ribosomal DNA. The first and second internal transcribed spacer (ITS‐1 and ITS‐2) regions of ribosomal DNA were amplified by polymerase chain reaction (PCR) from genomic DNA samples representing five species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella), denatured and then subjected to denaturing polyacrylamide gel electrophoresis (D‐PAGE) or single‐strand conformation polymorphism (SSCP) analysis. Differences in D‐PAGE profiles for both the ITS‐1 and ITS‐2 fragments (combined with an apparent lack of variation within individual species) enabled the unequivocal identification of the five species, and SSCP allowed the detection of population variation between some isolates representing E. acervulina, which remained undetected by D‐PAGE. The establishment of these approaches has important implications for controlling the purity of laboratory lines of Eimeria, for diagnosis and for studying the epidemiology of coccidiosis.

Genetic characterization of three unique operational taxonomic units of Eimeria from chickens in Australia based on nuclear spacer ribosomal DNA.

Coccidiosis of chickens is one of the commonest and economically most important parasitic diseases of poultry worldwide. Given the limitations of traditional approaches, molecular tools have been developed for the specific diagnosis of coccidiosis. Recently, a polymerase chain reaction (PCR)-based capillary electrophoresis (CE) method, employing genetic markers in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA, was established for both analytical and diagnostic purposes. The application of this method to investigate the epidemiology of coccidiosis and genetic structures of Eimeria populations on commercial chicken establishments has discovered genetic variants of Eimeria (i.e., new operational taxonomic units OTU-X, OTU-Y and OTU-Z) which were (based on CE analysis) distinct from those of species of Eimeria identified previously in chickens in Australia. The present characterization of these OTUs, based on their ITS-2 sequences and phylogenetic analyses of selected sequence data, provides first evidence to support that OTU-X represents a population variant of Eimeria maxima, and that OTU-Y and OTU-Z represent cryptic species of Eimeria. Further biological and genetic studies are needed to rigorously test these proposals and establish the specific status of these OTUs and their importance as pathogens in chickens. An understanding of the epidemiology of these population variants or cryptic species in Australia is central to designing and implementing effective vaccination and control strategies.

Investigating a persistent coccidiosis problem on a commercial broiler-breeder farm utilising PCR-coupled capillary electrophoresis.

In the present study, we utilised a polymerase-chain-reaction-coupled capillary electrophoresis (CE) approach to investigate the epidemiology of Eimeria species on a broiler–breeder farm in Victoria, Australia. The Eimeria populations of two flocks vaccinated against coccidiosis were followed over an 11-week period. All seven recognised Eimeria species of chickens were detected in both flocks. One flock suffered increased morbidity and mortality in its eighth week and had consistently higher Eimeria oocyst counts, species prevalences and rates of co-infections. Four Eimeria species included in the vaccine administered occurred at higher prevalences before the disease outbreak in the flock. Using the CE approach, two new, previously undescribed Eimeria genotypes were discovered in both chicken flocks, one of which dominated toward the end of the study period. The molecular approach proved versatile and capable of providing useful epidemiological data which could be used to investigate and interpret coccidiosis outbreaks.

Molecular separation of Oesophagostomum stephanostomum and Oesophagostomum bifurcum (Nematoda: Strongyloidea) from non-human primates.

The ITS-2 sequences for adult specimens of Oesophagostomum stephanostomum from the common chimpanzee and Oesophagostomum bifurcum from the Mona monkey were determined. For both species, the length and GC content of the ITS-2 sequences were 216 bp and 43%, respectively. While there was no unequivocal sequence difference among individual worms representing each of the two species, five (2.3%) interspecific nucleotide differences were detected. These differences were associated with the presence of unique restriction sites in the ITS-2 sequence of 0. stephanostomum for multiple endonucleases of diagnostic value for the differentiation of the two taxa by restriction analysis. Pairwise comparisons of the ITS-2 sequences of O. stephanostomum and O. bifurcum with published ITS-2 sequences for five different congeners indicated that these species from the subgenus Conoweberia are closely related, in accordance with previous morphological studies.

Genotyping Taenia tapeworms by single-strand conformation polymorphism of mitochondrial DNA

To overcome limitations in identifying tapeworms of the genus Taenia by traditional approaches, we have established a single‐strand conformation polymorphism (SSCP) method utilizing two different regions of mitochondrial (mt) DNA as targets. The NADH dehydrogenase 1 and the cytochrome c oxidase subunit I genes were amplified from genomic DNA by polymerase chain reaction (PCR), denatured and subjected to electrophoresis in mutation detection enhancement gels. SSCP analysis achieved delineation among eight different species of Taenia from different hosts based on characteristic profiles and enabled the detection of intraspecific variability in profiles for some taxa. This SSCP‐based typing method has important implications for taxonomy, diagnosis and for studying the genetic structure of Taenia populations.

Screening for nucleotide variations in ribosomal DNA arrays of Oesophagostomum bifurcum by polymerase chain reaction-coupled single-strand conformation polymorphism.

We exploited the high resolution capacity of polymerase chain reaction (PCR)‐coupled single‐strand conformation polymorphism (SSCP) to screen for sequence variation in the second internal transcribed spacer (ITS‐2) of ribosomal DNA (rDNA) among 77 individuals representing Oesophagostumum bifurcum from human or Mona monkey hosts from Africa. SSCP analysis revealed distinct profiles among some of the individuals, and sequence analysis of representative samples defined different ITS‐2 sequence types attributable to polymorphism at particular nucleotide positions. However, there was no unequivocal sequence difference between O. bifurcum individuals from humans and that from monkeys. This provided some support for the hypothesis that the parasite from the two hosts represents a single species and that the sequence microheterogeneity detected in the ITS‐2 rDNA region represents population variation, although the findings were insufficient to reject the proposal that the parasite represents different species. Overall, the results showed the usefulness of the SSCP‐sequencing approach for studying the genetic variation in O. bifurcum populations and indicated its potential to study macromolecular evolution and elucidate population differentiation at the molecular level.

A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species

Coccidiosis is a significant disease of poultry caused by different species of Eimeria. Differentiation of Eimeria species is important for the quality control of the live attenuated Eimeria vaccines derived from monospecific lines of Eimeria spp. In this study, high-resolution melting (HRM) curve analysis of the amplicons generated from the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2) was used to distinguish between seven pathogenic Eimeria species of chickens, and the results were compared with those obtained from the previously described technique, capillary electrophoresis. Using a series of known monospecific lines of Eimeria species, HRM curve analysis was shown to distinguish between Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella. Computerized analysis of the HRM curves and capillary electrophoresis profiles could detect the dominant species in several specimens containing different ratios of E. necatrix and E. maxima and of E. tenella and E. acervulina. The HRM curve analysis identified all of the mixtures as “variation” to the reference species, and also identified the minor species in some mixtures. Computerized HRM curve analysis also detected impurities in 21 possible different combinations of the seven Eimeria species. The PCR-based HRM curve analysis of the ITS-2 provides a powerful tool for the detection and identification of pure Eimeria species. The HRM curve analysis could also be used as a rapid tool in the quality assurance of Eimeria vaccine production to confirm the purity of the monospecific cell lines. The HRM curve analysis is rapid and reliable and can be performed in a single test tube in less than 3 h.