Detection of Hammondia heydorni-like organisms and their differentiation from Neospora caninum using random-amplified polymorphic DNA-polymerase chain reaction

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. New diagnostic genetic loci, based on random-amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), were developed to aid in the detection of H. heydorni-like parasites and to discriminate them from N. caninum and other related coccidians of dogs. On the basis of the data obtained from 5 random decamers, H. heydorni (Manhattan-1) and N. caninum (NC1) were characterized by distinct banding patterns (similarity index = 0.068). High-stringency PCR assays were developed from the sequences of 2 cloned bands (GenBank BZ592549 and BZ592593), uniquely amplified from H. heydorni. Interestingly, using these primers, PCR amplification was achieved only from 2 of the 5 isolates presumed to represent H. heydorni. The same result was obtained from these 5 isolates using a recently described PCR assay directed to the H. heydorni internal transcribed spacer-1. It is concluded that H. heydorni and N. caninum are genetically distinct and that such tools may be useful for more detailed characterization of the diversity of related parasites occurring in dogs.     

Molecular and biological characterization of Hammondia heydorni-like oocysts from a dog fed hearts from naturally infected white-tailed deer (Odocoileus Virginianus)

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. Although there is serological evidence of N. caninum infection in the white-tailed deer (Odocoileus virginianus), the parasite has not been yet isolated from the tissues of this host. In an attempt to isolate N. caninum from deer, hearts from 4 deer with antibodies to N. caninum were fed to 2 dogs. One of these dogs shed unsporulated oocysts 12-14 microm in diameter. Sporulated oocysts were not infective to Mongolian gerbils (Meriones ungulatus), and DNA isolated from these oocysts was not amplified using N. caninum-specific primers. However, positive amplification with the H. heydorni-specific first internal transcribed spacer (ITS-1) primers and common toxoplasmatiid ITS-1 primers confirmed the presence of H. heydorni DNA in the samples. The oocysts were considered to be H. heydorni on the basis of their morphology, biology, and molecular characteristics. This is the first record of a H. heydorni-like parasite in the white-tailed deer.     

犬MC1R基因R306ter与毛色性状相关性研究

目的　分析犬MC1R基因中R30 6ter位点多态性与犬毛色表型的相关性 ,为遗传育种 ,培育出更加优良的实验用犬奠定基础。方法　采用PCR SSCP技术 ,对MC1R基因R30 6ter位点进行基因多态性检测 ,分析位点多态性与毛色性状之间的相关性 ,并对该位点进行克隆测序。结果　PCR SSCP分析结果表明 ,R30 6ter位点序列具有多态性 ,表现为C、D二个等位基因和CC、CD及DD三种基因型。对R30 6ter多态性片段克隆测序发现 ,MC1R基因在编码第 30 6位氨基酸的密码子处存在一个由CGA到TGA的终止突变。结论　经统计分析结果表明在杂种犬中MC1R基因多态性与毛色性状不存在显著的相关性 ,这可能是由于外科手术学教学用犬是杂种犬 ,其遗传背景不同所致。由于MC1R基因的R30 6ter位点内存在碱基变异 ,因此在杂种犬中表现出明显的PCR SSCP多态性     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

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.     

Neospora caninum and Hammondia heydorni are separate species/organisms

Neospora caninum and Hammondia heydorni are two coccidian parasites with morphologically similar oocysts in canine feces. It was recently proposed that they are one species. In this paper, we review the biology and morphology of these parasites and present evidence that N. caninum and H. heydorni are separate species.     

Direct colony PCR-SSCP for detection of multiple pythiaceous oomycetes in environmental samples

Colony PCR was developed for detection of pythiaceous species recovered on selective agar plates without DNA extraction. A minute amount of mycelia from a single colony was picked up with a pipette tip and added directly to the PCR mix as template for DNA amplification. Successful amplification was achieved in over 95% of the colonies recovered from plant tissues, irrigation water and soil with species-specific primers or oomycete ITS-1 primers. PCR was inhibited in the case of colonies emerging from unwashed pine bark potting mix plates. Direct colony PCR with ITS-1 primers combined with single-strand conformation polymorphism analysis (SSCP) was used to determine population levels of single and multiple species in plant and environmental samples. Application of this technique for disease diagnosis and monitoring pathogen sources was explored, and the potential for studying diversity and population dynamics of other cultivated microbial communities in the environment is discussed.     

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.     

Single-strand conformational polymorphism analysis of the ribosomal internal transcribed spacer 1 for rapid species identification within the genus Pythium

Single-strand conformational polymorphism (SSCP) of the ribosomal internal transcribed spacer 1 (ITS-1) was characterized for 58 isolates of Pythium, representing 41 species from the five groups of Plaats-Niterink. Thirty-one species each produced a distinct SSCP pattern. Three species produced more than one unique pattern, corresponding to morphological subgrouping. The remaining seven species produced three distinct patterns with two or three morphologically similar species sharing a pattern. A successful blind test with four samples and the identification of eight previously unknown isolates from irrigation water demonstrated the reliability of this technique for species identification. Each SSCP pattern was defined and described by the positions of the top and bottom bands and the number of bands in between, which allows laboratories to use this technique without need to access the type isolates of Pythium species.     

用AFLP技术和16S rDNA PCR-RFLP分析毛苜蓿根瘤菌的遗传多样性

采用选择主增片断长度多态性（简称ＡＦＬＰ）ＤＮＡ指纹技术对采自我国云南省与西藏交界的高山地区的野生型豆科植物毛苜蓿根际土样分离的２９１株毛苜蓿（Ｍｅｄｉｃａｇｏ ｅｄｇｅｗｏｒｔｈｉｉ）根瘤菌进行遗传多样性的研究。从ＡＦＬＰ图谱中,揭示出毛苜蓿根瘤菌有较显著的遗传多样性,从２９１株中选择出９０年代表株用计算机进行树状图的分析。结果表明,所分析的菌株在７９％的相似性水平上聚类成３个群。对这９０年代表     

SNP frequency and allelic haplotype structure of Beta vulgaris expressed genes

Based on EST sequences, fragments of 37 genes have been amplified and sequenced in two inbred lines of sugar beet. The rate of single nucleotide polymorphisms (SNP) corresponded to 1 every 130 bp, with an average (nucleotide diversity) value of 7.6×10^–3. When extrapolated to the whole sugar beet genome, randomly compared lines differ at 5.4×10⁶ SNPs in the genetic pool considered. In a wider search for SNP-related polymorphisms, 96 fragments of expressed genes were scanned with SSCP (single-strand conformation polymorphism) and heteroduplex (HA) analyses in 8 inbred lines. One SSCP or HA polymorphism was found every 1,470 bp of amplified DNA, corresponding to 5×10⁵ SSCP or HA loci in the whole genome. This frequency, 11 times lower than the SNP rate, was attributed to the high frequency of base pair substitution along the amplified fragment analysed electrophoretically. Therefore nucleotide variability was further studied by sequencing fragments of 10 genes in the same 8 lines. The results indicate that sugar beet alleles of expressed genes are very frequently organized as robust intragene haplotypes. In the 8 lines analysed, two haplotypes were identified for each of three gene fragments, three haplotypes for six gene fragments and four haplotypes for one gene fragment which is in good correspondence with the number of alleles detected by SSCP and HA analysis. In a cross between two lines, SSCP or HA alleles of expressed genes have 54% probability to be different.     

Single strand conformation polymorphism is a sensitive method for screening nucleotide variations in Mycosphaerella graminicola

Single Strand Conformation Polymorphism (SSCP) and sequencing were performed in order to assess molecular polymorphism of mating type sequences in the heterothallic ascomycete Mycosphaerella graminicola, the causal agent of Septoria tritici blotch of wheat. The screening was undertaken on mat1-1 and mat1-2 partial sequences of 341 and 657 bp, respectively, amplified with multiplex PCR from 510 French single-conidial strains plus the two reference isolates IPO323 and IPO94269 from The Netherlands. After restriction with Taq1 in order to reduce the fragment sizes, all digested amplicons were subjected to SSCP. Sequencing was then performed when a SSCP pattern deviates from the most frequently occurring profile. Among the assessed strains, 228 ones plus IPO323 were MAT1-1 and 282 ones plus IPO94269 were MAT1-2. Among the MAT1-1 strains, only a single one exhibited a SSCP profile distinct to the other MAT1-1 strains, whereas 10 MAT1-2 strains (among which 2 and 4 with same profiles, respectively) showed a SSCP profile differing to the other MAT1-2 strains. Sequencing revealed that all polymorphisms observed on SSCP gels were single nucleotide variations and all strains displaying the same SSCP profiles showed identical nucleotide sequences. Among the seven disclosed nucleotide variations, only two were non-synonymous and both were non-conservative. This study reports a high sensitivity of SSCP allowing detection of single point mutations in M. graminicola, shows a conservation of mating type idiomorphs in the fungus at both sequence and population scales, but also suggests a difference in polymorphism level between the two mating type sequences.     

Differential identification of mycoplasma pulmonis and M. arthritidis using PCR-based RFLP.

Mycoplasma pulmonis and Mycoplasma arthritidis were differentially identified using PCR-restriction fragment length polymorphism (RFLP). A genus-specific sequence of mycoplasma was amplified by PCR and the PCR products were digested with the restriction enzyme SmaI. Each PCR product from the four isolates of M. pulmonis was digested with SmaI into two fragments; however, there was no digestion in the PCR product from M. arthritidis. This method might be useful to differentiate infection of M. pulmonis from that of M. arthritidis.     

Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis

AIMS: To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. METHODS AND RESULTS: By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. CONCLUSIONS: PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.     

Concordant evolution of mitochondrial and nuclear genomes in the wheat pathogen Phaeosphaeria nodorum

We compared patterns of mitochondrial restriction fragment length polymorphism (RFLP) diversity with patterns of nuclear RFLP diversity to investigate the effects of selection, gene flow, and sexual reproduction on the population genetic structure and evolutionary history of the wheat pathogen Phaeosphaeria nodorum. A total of 315 fungal isolates from Texas, Oregon, and Switzerland were analyzed using seven nuclear RFLP probes that hybridized to discrete loci and purified mitochondrial DNA that hybridized to the entire mtDNA genome. Forty-two different mitochondrial haplotypes and 298 different nuclear haplotypes were detected. The two most frequent mtDNA haplotypes were present in every population and represented 32% of all isolates. High levels of gene flow, low levels of population subdivision, no evidence for either host specificity or cyto-nuclear disequilibrium were inferred from the analysis of both genomes. The concordance in estimates of these population genetic parameters from both genomes suggests that the two genomes experienced similar degrees of migration, genetic drift and selection.     

Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis

The genetic structure of Mycosphaerella fijiensis populations around the world was examined using DNA restriction fragment length polymorphism (RFLP) markers. Allele frequencies at 19 nuclear RFLP loci were estimated in a sample of 136 M. fijiensis isolates from five geographical populations representative of banana-producing areas (South-East Asia including the Philippines and Papua New Guinea, Africa, Latin America and Pacific Islands). Within each population, gametic disequilibrium tests between the 19 nuclear RFLP loci were mainly non significant ( P > 0.05), indicating that random sexual reproduction occurred in these populations. All M. fijiensis populations had a high level of genotypic and allelic diversity ( H , gene diversity: 0.25–0.59). The highest levels of gene diversity were found in the two South-East Asian populations ( H : 0.57 and 0.59). Most of the alleles (> 88%) detected in Africa, Latin America and Pacific Islands populations were also detected in South-East Asian populations. Furthermore, a high and significant ( P < 0.05) level of genetic differentiation was observed among M. fijiensis geographical populations (overall estimate of F_st : 0.32). These results were consistent with the hypothesis that M. fijiensis originated in South-East Asia and spread recently to other parts of the world. The level of allelic diversity in M. fijiensis populations from regions other than South-East Asia was drastically reduced, indicating founder effects. The data also suggested rare occurrence of migration of M. fijiensis between continents.     

Hammondia heydorni from the Arabian mountain gazelle and red fox in Saudi Arabia

Unsporulated oocysts were detected in the feces of an Arabian red fox (Vulpes vulpes arabica) between 6 and 8 days after it had been fed meat from Arabian mountain gazelles (Gazella gazella) known to contain sarcocysts. No oocysts were discovered in the feces of other experimental cubs, although sporocysts of Sarcocystis spp. were passed subsequently by all cubs that were fed gazelle meat, including those fed with reem (G. subgutturosa marica). The oocysts sporulated in 3 days at room temperature (25 +/- 2 C); they were 10.9 +/- 1.4 x 10.1 +/- 1.3 microm, with 2 sporocysts measuring 6.0 +/- 0.6 x 4.7 +/- 0.8 microm, each with 4 sporozoites. Sporulated oocysts were identified as those of Hammondia heydorni using molecular and standard morphometric techniques. Sequence differences between 2 fox and 3 dog isolates of H. heydorni were detected and allowed differentiation between the 2 populations of the organism. The involvement of Neospora caninum was excluded using molecular methods. The Arabian red fox and the Arabian mountain gazelle in Saudi Arabia are new, definitive and intermediate hosts for H. heydorni.     

Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strand conformation polymorphism analysis of the coat protein gene

Citrus tristeza closterovirus (CTV) isolates of several geographical origins were compared for variations in their coat protein (CP) gene by analysis of single-strand conformation polymorphism (SSCP). The CP gene of 17 isolates was reverse transcribed, amplified by polymerase chain reaction (PCR), and 22 clones were inserted into a plasmid vector. These clones were sequenced and found to have between 91.7% and 99.8% sequence homology. Clones were amplified and the PCR products denatured and compared by SSCP analysis in 8% polyacrylamide gels. Using two different electrophoretic conditions, the patterns were different for 16 or 17 clones. Four pairs of clones (T36/T66, P1/Q2, 03/8Q, and E1/E2) differing by 10, 2, 1 and 1 nucleotides, respectively, could not be distinguished using either condition. When these clones were compared by SSCP after digestion with Eco91I (BstEII) three of the pairs (T36/T66, P1/Q2, and 03/8Q) could be differentiated, whereas the clones E1 and E2 (differing by 1 nucleotide) remained indistinguishable. Thus, SSCP analysis combining two electrophoretic conditions and restriction of eight clones with Eco91I allowed discrimination between 21 of the 22 CP gene clones selected. SSCP analysis may provide a procedure to identify and differentiate CTV isolates based on comparisons of several genes or gene regions. It is rapid and cheap and may drastically reduce the amount of sequencing necessary for accurate comparisons.