Paul A. Horgen

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

ABSTRACT The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

Real-Time PCR Assay for Detection and Genotype Differentiation of Giardia lamblia in Stool Specimens

ABSTRACT Real-time PCR, using dual-labeled fluorescent probes targeting the β-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.

The mitochondrial DNAs of Agaricus: heterogeneity in A. bitorquis and homogeneity in A. brunnescens

SummaryMitochondrial DNAs were isolated from four cultivated strains of the commercial two-spored mushroom Agaricus brunnescens (bisporus) and from ten isolates of the four spored mushroom Agaricus bitorquis. Digestion of the fungal mitochondrial DNA with restriction endonucleases yielded numerous fragments. Summation of the fragment sizes gave a mitochondrial DNA size of 98.3 ± 2.4 kilobases (kb) (64.9 x 106 daltons) for A. brunnescens. The size of the mitochondrial DNA ranged from 148.5 ± 10.8 kb (98.0 x 106 daltons) to 176.3 ± 12.0 kb (116.4 x 106 daltons) for A. bitorquis. The restriction patterns, produced by a variety of endonucleases, were identical for all four isolates of A. brunnescens. The ten isolates of A. bitorquis demonstrated extensive restriction pattern heterogeneity and have been tentatively assigned into four groups. Approximately 60% of the A. bitorquis mitochondrial DNA restriction fragments show sequence homology with A. brunnescens mitochondrial DNA based on DNA — DNA hybridizations.

Isolation and characterization of the cerato-ulmin toxin gene of the Dutch elm disease pathogen, Ophiostoma ulmi

The hydrophobic protein cerato-ulmin (CU), produced by Ophiostoma ulmi, has been implicated in the pathogencity of this fungus on elm. Primers were designed based on the nucleotide sequence deduced from the published CU amino-acid sequence, and a DNA fragment of the cu gene was amplified using the polymerase chain reaction. The amplified cu fragment was used as a hybridization probe to identify and isolate the cu gene from a genomic DNA library of an aggressive isolate of O. ulmi (= O. novo-ulmi). The cu coding region is interrupted by two introns and encodes a 100 amino-acid prepro-CU polypeptide that is processed to a 75 amino-acid mature protein upon secretion. CU shows significant sequence similarity to hydrophobins secreted by certain other fungi.

Homology between mitochondrial DNA of Agaricus bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis

SummaryAgaricus bisporus, the cultivated mushroom, contains a mitochondrial fragment (50H) which was previously demonstrated by Southern hybridization to have sequence similarity to an internal region of pEM, a linear mitochondrial plasmid of Agaricus bitorquis. The nucleotide sequence of 50H was determined and compared to the sequence of the corresponding pEM fragment. The region of sequence homology on pEM is contained within an open reading frame (ORF) that may encode an RNA polymerase, but 50H is neither an intact nor a complete copy of the ORF. pEM also contains an ORF with characteristics of genes for virus-encoded DNA polymerases. pEM appears to be very similar to other linear mitochondrial plasmids (in fungi and higher plants) reported to contain ORFs that may encode the same types of polymerases. The potential functionality of the pEM sequence suggests that it has diverged less than the mitochondrial fragment from a common ancestor.

Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease

SummaryA transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 × 103 transformants/μg DNA per 107 protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.

Physical mapping of the mitochondrial genome of the cultivated mushroom Agaricus brunnescens (- A. bisporus)

SummaryMitochondrial (mt) DNA from the commercial mushroom Agaricus brunnescens Peck [= A. bisporus (Lange) Imbach] was purified by cesium chloride/bisbenzimide gradient centrifugation. A physical map of the mtDNA fragments produced by BamHI, EcoRl, and PvuII digestion was generated by filter hybridizations with radiolabelled BamHI mtDNA probes. The A. brunnescens mtDNA was a circular molecule 136 kilo-basepairs (kbp) in length and contained an inverted repeat between 4.6 and 9.2 kbp in size. Orientational isomers of the mitochondrial genome were not detected. The positions of six genes were located on the A. brunnescens mtDNA map by heterologous hybridization. No coding function has yet been ascribed to the inverted repeat. The large rRNA gene was located on the smaller single copy region. The genes for cytochrome b, cytochrome oxidase (subunit III), ATPase (subunits 8 and 6) and the small rRNA were located on different regions of the larger single copy region.

The indigenous coastal Californian population of the mushroom Agaricus bisporus, a cultivated species, may be at risk of extinction

This study employed nuclear and mitochondrial markers to assess the present‐day composition of the population of Agaricus bisporus in coastal California. Favourable weather in the fall, winter and spring of 1990–91 furnished an uncommon opportunity to collect and study field material of the ‘button mushroom’A. bisporus, a cultivated species, from the region. The previous such season occurred 13 years earlier. Ninety‐five nonredundant cultures from field material were prepared and genotypically characterized. These data were combined with data from earlier studies. Multilocus nuclear and mitochondrial genotypes were determined for 123 individuals. Genotypes were compared in pairwise fashion both within the sample and between this sample and others of diverse geographical origin or commercial provenance. Using parametric analysis and cluster analysis of nuclear similarities, and also mitochondrial data, two elements – indigenous and European – were apparent within the sample. This was consistent with our earlier results on a much smaller sample. At least 10 mitochondrial haplotypes (MTs) were present; based on genotypic similarities of associated nuclei, five (or six) MTs were Californian, four were European, and one was ambiguous. Based on MT origins, 54% of the 121 classifiable individuals in California were of European ancestry; natives constituted a minority at 46%. Even in the indigenous Monterey cypress habitat, where 84% of all individuals from California were sampled, non‐native A. bisporus appeared to have achieved parity (at 48–49%) with the native population. In all other habitats, which are far more extensive, European individuals outnumbered Californian natives by 4:1. Some evidence of hybridization between the two ancestral groups was found. European strains appear to have been resident in California for approximately one century. The extensive occupancy of the native habitat by non‐native germ plasm, the apparent inability of native strains to occupy or compete in non‐native habitats, and the disproportionately large inoculum reservoirs represented by non‐native habitat and agronomic activities all suggest that the native population is under considerable competitive pressure in what may be a very dynamic situation. If this surmise is correct, the native population may be at serious risk of further contraction, irreversible dilution through interbreeding, and possibly even extinction.

Mitochondrial DNA variation in natural populations of the mushroom Agaricus bisporus

We investigated the patterns of mitochondrial DNA variation in the global population of the commercial mushroom Agaricus bisporus. Through the analysis of RFLPs among 441 isolates from nine countries in North America and Eurasia, we found a total of 140 mtDNA haplotypes. Based on population genetic analysis, there are four genetically distinct natural populations in this species, found in coastal California, desert California, France and Alberta (Canada). While 134 of the 140 mtDNA haplotypes were unique to single geographical regions, two mtDNA haplotypes, mt001 and mt002, were found in almost every population surveyed. These two mtDNA haplotypes also predominate among cultivars used throughout the world for at least the last two decades. These two mtDNA haplotypes are more similar to the cosmopolitan groups of mtDNA haplotypes than to the indigeneous clusters of mtDNA haplotypes from the two Californian regions.

Strategies for the efficient recovery of Agaricus bisporus homokaryons

Genetic analysis and selective breeding of cultivated mushrooms require the isolation of homokaryons from heterokaryotic stocks. This is problematic for Agaricus bisporus (Lange) Imbach (= ? A. brunnescens Pk.) because homokaryons are uncommon, among both single spore isolates (SSIs) and protoplast regenerates (PRs) from heterokaryons, and because there is no directly observable criterion such as the presence or absence of clamp connections that would permit the reliable sorting of such isolates by their effective ploidy level (IN vs N+N). As a result of such difficulties, progress in these areas has been greatly retarded. A capsule review of approaches to solving this problem provides a context for our study in which several alternative ploidy screening criteria were evaluated. Prior studies have relied upon the following criteria: 1) Colony morphology: this may be appropriate following deheterokaryotization (Dickhardt, 1985) but may be inappropriate for sorting diverse meiotic offspring. [However, see Kligman (1943) and Pelham (1967).] 2) Growth rate: limited published speculation and evidence (e.g., Pelham, 1967; Elliott, 1972; Castle et al., 1987) and widespread industrial practice (M. Spear, pers. comm.; K. Dahlberg, pers. comm.) suggest that homokaryons may grow more slowly than heterokaryons, but hard data and analysis concerning this trait remain unavailable. 3) Auxotrophy: the reported Mendelian behavior of auxotrophic mutants (Raper et al., 1972) leads to the expectation that uncomplemented homokaryotic offspring (and some heterokaryotic offspring) of auxotrophic parentage will be auxo-