Microsatellite markers reveal genetic differentiation among populations of Sclerotinia sclerotiorum from Australian canola fields

Eight microsatellite markers were applied to 154 Sclerotinia sclerotiorum isolates from four Australian canola fields, to determine the extent of genetic variation and differentiation in populations of this pathogen. A total of 82 different haplotypes were identified and in each population many haplotypes were unique. Mycelial compatibility grouping, a phenotypic marker system controlled by multiple loci, was often associated with groups of identical or closely related microsatellite haplotypes. Genotypic diversity ranged from 36% to 80% of maximum in the four populations, and gene diversity ranged from 0.23 to 0.79. Genotypic disequilibrium analyses on each of the four populations suggested that both clonal and sexual reproduction contributed to population structure. Analyses based on genetic diversity and fixation indices demonstrated a moderate to high level of differentiation (R_ST=0.16–0.33, F_ST=0.18–0.23) between populations from New South Wales and those from Victoria. Despite this genetic diversity, most isolates did not vary in virulence on canola leaves.     

Evidence for outcrossing in Phytophthora sojae and linkage of a DNA marker to two avirulence genes

Two genetically different isolates of the homothallic Oomycete, Phytophthora sojae, were demonstrated to outcross and form hybrid oospores after co-culturing in vitro. Random amplified polymorphic DNA (RAPD) markers revealed ten hybrids among 354 oospores analysed. One F₁ hybrid was allowed to self fertilise and produce an F₂ population of 247 individuals. Among 53 F₂ individuals, selected at random, 18 polymorphic RAPD markers were observed to segregate at near 3:1 Mendelian ratios, consistent with segregation for dominant alleles at single loci. Segregation of virulence against soybean resistance genes Rps1a, 3a, and 5 revealed that the avirulence genes Avr1a, 3a and 5 were dominant to virulence. Avirulence against these three resistance genes appeared to be conditioned by one locus for Avr1a and two independent, complementary dominant loci for both Avr3a and Avr5. Segregation of virulence against Rps6 was in the ratio of 1:2:1 (avirulent:mixed reaction:virulent), suggesting a semi-dominant allele at a single locus. Two avirulence genes and one RAPD marker formed one linkage group, in the order Avr3a, OPH4-1, Avr5, each separated by approximately 5 cM. Our results confirm that outcrossing occurred between the parental isolates, and that sexual recombination under field conditions may play an important role in generating and maintaining genetic diversity in populations of P. sojae.     

Genetic structure of a population of the fungus Leptosphaeria maculans in a disease nursery of Brassica napus in Australia

Microsatellite, minisatellite and mating type markers were used to determine the genetic structure of the fungus Leptosphaeria maculans within a disease nursery, where Brassica napus lines were screened for resistance to blackleg disease under high inoculum pressure. Fungal isolates were collected from pseudothecia in infected stubble and pycnidia within cotyledon lesions on seedlings within the nursery. Genetic diversity was high with gene diversity at H=0.700 across four polymorphic loci, and genotypic diversity at D=0.993. Among the 159 isolates analysed, 102 multilocus genotypes were identified. The even distribution of mating type idiomorphs MAT1-1 and MAT1-2 and gametic equilibrium within the population provided further evidence of random mating. Genetic diversity was distributed on a very fine scale in the disease nursery. The majority of genetic diversity (67%) was distributed among conidia within a lesion or among ascospores from a piece of stubble, while the remainder (33%) was distributed within lesions on seedlings or different stubble pieces. There were no among-group differences between samples from stubble and seedlings. This is consistent with the low level of genetic differentiation between the ascospore and conidia samples (F _ST=0.017) indicating that all isolates of L. maculans from the disease nursery most likely belong to one population, and that ascospores form the primary inoculum in the disease nursery.     

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.     

DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

Summary The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)₄, (GTG)₅, (CA)₈, and (TCC)₅, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.     

Identification and characterization of 31 isolates of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> (Spirochaetales,Spirochaetaceae) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes ricinus (L., 1758) ticks. The aim of our study was to identify spirochaete isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their characterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infraorder Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.     

Homologous linear plasmids in mitochondria of three species of wheat bunt fungi,Tilletia caries,T. laevis and T. controversa

Summary All isolates of Tilletia spp. investigated (five isolates of T. caries, including one from Japan, two isolates of T. laevis, and five isolates of T. controversa) contained a linear DNA plasmid ranging in size from 7.2 to 7.6 kb. All plasmids were highly homologous to each other as shown by DNA-DNA hybridization and comparison of restriction enzyme sites. Variability in the size of the plasmid was found to be due to differences within a central region of the plasmid. No homology between the plasmid and mitochondrial or nuclear DNA was found, but the mitochondrial origin of the plasmid was confirmed.     

Characterization of mutations in the two-component histidine kinase gene AbNIK1 from Alternaria brassicicola that confer high dicarboximide and phenylpyrrole resistance

Highly iprodione- and fludioxonil-resistant field and laboratory isolates of A. brassicicola were found to be either moderately sensitive or tolerant to osmotic stress. AbNIK1, a two-component histidine kinase gene, was isolated from a fungicide-sensitive strain. The predicted protein possessed the six tandem amino acid repeats at the N-terminal end, which is a landmark of osmosensor histidine kinases from filamentous fungi. A comparison of the nucleic acid sequences of the AbNIK1 gene from fungicide-sensitive and fungicide-resistant isolates revealed the presence of mutations in six of the seven resistant strains analyzed. Null mutants were all found to be moderately sensitive to osmotic stress, indicating that they are similar to Neurospora crassa Type I os-1 mutants. Only one mutation, corresponding to a single amino acid change within the H-box of the kinase domain, was found in an osmotolerant strain. These results suggest that AbNIK1p participates in osmoregulation and that expression of the fully functional enzyme is essential for dicarboximide and phenylpyrrole antifungal activities.     

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio – Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n = 20), Typhimurium (n = 4), Zanzibar (n = 2), Manhattan (n = 1), Bovismorbificans (n = 1), Amsterdam (n = 1), Saint Paul (n = 1), Kentucky (n = 1) and Muenster (n = 1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1–5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.     

Genetic structure and variability of virus populations in cross-protected grapevines superinfected by Grapevine fanleaf virus

Recombination was assessed in a vineyard site in which grapevines cross-protected with mild strains GHu of Grapevine fanleaf virus (GFLV) or Ta of Arabis mosaic virus (ArMV) were superinfected with GFLV field isolates following transmission by the nematode vector Xiphinema index. The genetic structure and variability within RNA2 of isolates from grapevines co-infected with GFLV field isolates and either GFLV-GHu or ArMV-Ta were characterized to identify intra- and interspecies recombinants. Sequence analysis and phylogenetic relationships inferred intraspecies recombination among GFLV field isolates but not between field isolates and GFLV-GHu. SISCAN analysis confirmed a mosaic structure for two GFLV field isolates for which recombination sites were located in the movement protein and coat protein genes. One of the recombinants was found in eight grapevines that were in close spatial proximity within the vineyard site, suggesting its transmission by X. index. No interspecies recombination was detected between GFLV field isolates and ArMV-Ta. Altogether, our findings suggest that mild protective strains GFLV-GHu and ArMV-Ta did not assist the emergence of viable recombinants to detectable level during a 12-year cross-protection trial. To our knowledge, this is the first extensive characterization of the genetic structure and variability of virus isolates in cross-protected plants.     

Population Structure and Genetic Diversity within California Citrus tristeza virus (CTV) Isolates

The Closterovirus, Citrus tristeza virus (CTV) is an aphid-borne RNA virus that is the causal agent of important worldwide economic losses in citrus. Biological and molecular variation has been observed for many CTV isolates. In this work we detected and analyzed sequence variants (haplotypes) within individual CTV isolates. We studied the population structure of five California CTV isolates by single strand conformation polymorphism (SSCP) analysis of four CTV genomic regions. Also, we estimated the genetic diversity within and between isolates by analysis of haplotype nucleotide sequences. Most CTV isolates were composed of a population of genetically related variants (haplotypes), one being predominant. However in one case, we found a high nucleotide divergence between haplotypes of the same isolate. Comparison of these haplotypes with those from other isolates suggests that some CTV isolates could have arisen as result of a mixed infection of two divergent isolates.     

Staphylococcus aureus as source of catheter‐related bloodstream infection evaluated by PFGE and rep‐PCR typing in a Brazilian hospital

Staphylococci are a common cause of catheter‐related bloodstream infection (CR‐BSI), and epidemiological typing is an important tool for effective infection control. This study evaluated by PFGE and rep‐PCR whether Staphylococcus aureus strains isolated from skin and catheter tips were related to specimens isolated from blood. A prospective observational study, carried out in a clinical surgical ward at a Brazilian hospital between September 2000 and November 2002, investigated non‐tunneled central venous catheters from 179 patients. S. aureus isolates were mainly obtained from blood (41.4%), while coagulase‐negative staphylococci strains were more often isolated from the skin at the catheter insertion site (49.7%) and from the catheter tip (57.5%). Among the 21 strains isolated from 9 patients at 2 or 3 sites simultaneously, 9 were methicillin‐resistant S. aureus (MRSA) and 12 were methicillin‐susceptible S. aureus (MSSA). Seven patients harbored the same S. aureus strain isolated from the skin, blood and/or catheter tip cultures. MRSA isolates belonged to one PFGE pattern (type A‐ subtypes A₁, A₂ and A₃), and to two rep‐PCR patterns (a and b). MSSA isolates were distinguished in five PFGE (B to F) and in three rep‐PCR (c, d and e) patterns. Both PFGE and rep‐PCR methods indicated that the skin at the catheter insertion site was the origin of CR‐BSI caused by S. aureus.     

Studies on the presence and persistence of Pasteurella multocida serovars and genotypes in fowl cholera outbreaks

Pasteurella multocida was isolated from poultry on six farms (one free-range duck farm, one free-range turkey farm, one conventional enclosed turkey farm, and three free-range layer farms) suffering fowl cholera outbreaks. In addition, historical isolates from previous outbreaks were available for the conventional turkey farm and the three free-range layer farms. The isolates were serotyped using the Heddleston scheme and genotyped using multi-locus sequencing typing. In the current outbreaks, two of the farms had two different sequence types (STs) of P. multocida in the investigated outbreak (the free-range turkey farm and one of the free-range layer farms). The remaining four farms had one ST within the investigated outbreak. In looking at the historical isolates, two of the four farms had multiple genotypes involved. On the four farms with historical isolates from previous outbreaks, at least one new genotype was present in the investigated outbreak as compared with the historical isolates. On one layer farm, one genotype persisted over a 10-year period. Serotyping revealed the presence of multiple serovars in the current and historical outbreaks, with serovars sometimes changing over time. This study has shown that several STs of P. multocida can be present during some outbreaks of fowl cholera, although other outbreaks involve a single ST. Also, the STs present on a property suffering repeated fowl cholera can both persist and change over time.     

Sequence variability and phylogenetic relationship of betasatellite isolates associated with yellow vein mosaic disease of mesta in India

Six betasatellite isolates associated with the yellow vein mosaic disease in mesta crops grown under three different geographical locations of India have been characterized. These six isolates and the one previously reported from eastern India could be divided into two distinct Types. The first Type, consisted of four betasatellite isolates characterized from northern and southern regions of India, was observed to be the newer isolates of Ludwigia leaf distortion betasatellite. The second Type, comprised three betasatellite isolates obtained from the eastern part of India, showed highest sequence identity with Cotton leaf curl Multan betasatellite and appeared to be the newer isolates of it. These isolates present within each of these two betasatellite species showed limited variability with respect to their individual group. The results thus indicated the association of two different betasatellite species with yellow vein mosaic disease of mesta in India and highlighted the possible adaptation of mesta crops as a newer hosts by these two betasatellite species.     

Phylogenetic relationships of unclassified, satellitic Pasteurellaceae obtained from different species of birds as demonstrated by 16S rRNA gene sequence comparison

Avian haemophili demonstrating in vitro satellitic growth, also referred to as the V-factor or NAD requirement, have mainly been classified with Avibacterium paragallinarum (Haemophilus paragallinarum), Avibacterium avium (Pasteurella avium), Avibacterium volantium (Pasteurella volantium) and Avibacterium sp. A (Pasteurella species A). The aim of the present study was to assess the taxonomic position of 18 V-factor-requiring isolates of unclassified Haemophilus-like organisms isolated from galliforme, anseriforme, columbiforme and gruiforme birds as well as kestrels and psittacine birds including budgerigars by conventional phenotypic tests and 16S rRNA gene sequencing. All isolates shared phenotypical characteristics which allowed classification with Pasteurellaceae. Haemolysis of bovine red blood cells was negative. Haemin (X-factor) was not required for growth. Maximum-likelihood phylogenetic analysis including bootstrap analysis showed that six isolates were related to the avian 16S rRNA group and were classified as Avibacterium according to 16S rRNA sequence analysis. Surprisingly, the other 12 isolates were unrelated to Avibacterium. Two isolates were unrelated to any of the known 16S rRNA groups of Pasteurellaceae. Two isolates were related to Volucribacter of the avian 16S rRNA group. Seven isolates belonged to the Testudinis 16S rRNA group and out of these, two isolates were closely related to taxa 14 and 32 of Bisgaard, whereas four other isolates were found to form a genus-like group distantly related to taxon 40 and one isolate remained distantly related to other members of the Testudinis group. One isolate was closely related to taxon 26 (a member of Actinobacillus sensu stricto). The study documented major genetic diversity among V-factor-requiring avian isolates beyond the traditional interpretation that they only belong to Avibacterium, underlining the limited value of satellitic growth for identification of avian members of Pasteurellaceae. Our study also emphasized that these organisms will never be isolated without the use of special media satisfying the V-factor requirement.     

Genotype and haplotype analysis of cell cycle genes in sporadic colorectal cancer in the Czech Republic

The Czech Republic has one of the highest incidences of colorectal cancer (CRC) in the world. To assess the role of genetic variants on the disease, we genotyped polymorphisms in the TP53 (rs17878362:A₁>A₂, rs1042522:G>C, rs12947788:C>T, and rs17884306:G>A), CDKN1A (rs1801270:C>A and rs1059234:C>T), and CDKN2A (rs3731249:G>A, rs11515:C>G, and rs3088440:C>T) genes in 614 hospital‐based CRC cases and 614 matched controls from the country. Despite the tendency toward differential distribution of variant allele frequencies for some polymorphisms, none was significantly associated with CRC risk. We observed differential distribution of major haplotypes arising from four polymorphisms in the TP53 gene between cases and controls (global P<0.0001). The two most common haplotypes, A₁GCG and A₂CCG, were present in 81% of the cases compared to 71% of the controls. In comparison to the most common haplotype (A₁GCG), the haplotype A₂CCG was associated with an increased risk (odds ratio [OR], 1.40; 95% confidence interval [CI], 1.07–1.82), while the four other haplotypes A₁CCG (OR, 0.60; 95% CI, 0.45–0.79), A₂GCG (OR, 0.53; 95% CI, 0.35–0.81), A₁GTG (OR, 0.31; 95% CI, 0.15–0.64), and A₁GCA (OR, 0.19; 95% CI, 0.07–0.51) were associated with a decreased risk. The effect of haplotypes in the TP53 gene was similar in colon (global P<0.0001) and rectal cancers (P=0.006). No association with the disease was observed with haplotypes of the CDKN1A and CDKN2A polymorphisms. The results from this study suggest that prevalent haplotypes within the TP53 gene may modulate CRC risks in the population. Hum Mutat 0, 1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Low‐level resistance and clonal diversity of Pseudomonas aeruginosa among chronically colonized cystic fibrosis patients

A prospective study was conducted in Brazil to evaluate antimicrobial resistance patterns and molecular epidemiology of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients with chronic lung infection. All isolates were obtained between May 2009 and June 2010 from 75 patients seen in four reference centers in Brazil: HCPA (20 patients) and HEOM (15 patients), located in southern and northeastern Brazil, respectively; IFF (20 patients) and HUPE (20 patients), both in southwestern Brazil. Antimicrobial susceptibility testing, PCR for detection of carpapenemases, and pulsed‐field gel electrophoresis (PFGE) were performed in 274 isolates. A total of 224 PFGE types were identified and no clones were found circulating among the centers or within the same center. Despite the chronic infection, most patients were colonized by intermittent clones. Only three patients (4%) maintained the same clone during the study. The resistance rates were lower than 30% for the majority of antimicrobials tested in all centers and only 17% of isolates were multiresistant. Isolates (n = 54) with reduced susceptibility to imipenem and/or meropenem presented negative results for bla_SPM‐1, bla_IMP?1, bla_VIM, and bla_KPCgenes. Our results indicate an unexpected low level of antimicrobial resistance and a high genotypic diversity among P. aeruginosa from Brazilian chronic CF patients.     

Outcrossing in the homothallic oomycete,Pythium ultimum,detected with molecular markers

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F₁ yielded a F₂ population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.     

The effect of anesthesia on the photoresponses of four sympatric species ofDrosophila

Five isofemale strains from each of four sympatric species,Drosophila immigrans, D. repleta, D. melanogaster, andD. affinis, were lightly anesthetized with ether, CO₂, Flynap, or cold temperature. The photoresponse of each treatment group was measured in an open field and a Y-tube apparatus. Relative light values were compared with those of the unanesthetized control groups of each species, as well as among the different treatments themselves and among species. Significant differences between species were apparent for all treatments in the Y-tube and for all treatments except ether and CO₂ in the open field. Response to anesthesia may differ between species due, in part, to their differing genotypes. Comparing treatment groups within species in the Y-tube design, all species exceptD. melanogaster showed significant differences between treatments. In the open field, onlyD. affinis was significantly affected. Particularly in the Y-tube, anesthetized flies generally differed significantly from the controls, indicating that the use of anesthesia during a behavioral study could cause deviations in behavior from that of normal unanesthetized flies.     

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary

Ornithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A–E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.