Biodiversity of Clostridium botulinum Type E Strains Isolated from Fish and Fishery Products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Prevalence of Clostridium botulinum in Finnish Trout Farms: Pulsed-Field Gel Electrophoresis Typing Reveals Extensive Genetic Diversity among Type E Isolates

The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg⁻¹, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.     

Molecular and genetic analyses of geographic variation in isolates of Phoma macrostoma used for biological weed control

Molecular and genetic approaches were used to evaluate the genetic relatedness among isolates of the fungus Phoma macrostoma Montagne originating from Canada and Europe and to other species in the genus Phoma. Distinct differences were observed in genetic variation among nine species of the genus Phoma. Randomly amplified polymorphic DNA (RAPD) revealed the presence of intraspecific genetic variation among the isolates of P. macrostoma, with the isolates being used for biological weed control being distributed in a distinct phylogenetic cluster. Additional variation within the biocontrol isolate cluster in P. macrostoma was revealed by pulsed field gel electrophoresis (PFGE), which showed that biocontrol isolates generated two different chromosomal profiles, however the profiles did not relate to their Canadian ecozone origin. Mating studies showed that biocontrol isolates of P. macrostoma from Canada did not produce sexual reproductive structures and were incapable of crossing. These studies also confirmed that no obvious differentiation exists among the biocontrol isolates of P. macrostoma from Canadian Ecozones 3 and 4.     

Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes

In order to determine a possible genomic divergence of Erwinia amylovora'fruit tree' and raspberry strains from North America, several isolates were differentiated by pulsed-field gel electrophoresis (PFGE) analysis, the size of short DNA sequence repeats (SSRs) and the nucleotide and deduced amino acid sequences of their hrpN genes. By PFGE analysis European strains are highly related, whereas strains from North America were diverse and were further distinguished by the SSR numbers from plasmid pEA29. The E. amylovora strains from Europe showed identical HrpN sequences in contrast to the American isolates from fruit trees and raspberry. Those were related to each other, but distinguishable by their HrpN patterns. The Asian pear pathogens differed in HrpN among each other and from E. amylovora. Erwinia pyrifoliae isolates and the Erwinia strains from Japan were ordered via their HrpN sequences in agreement with the PFGE patterns. For all three pathogens, dendrograms from PFGE and sequence data indicate an evolutionary diversity within the species in spite of a genetic conservation for parts of the hrpN genes suggesting a long persistence of the Asian pear pathogens in Korea and Japan as well as of fire blight in North America. Some of the divergent American E. amylovora isolates share PFGE patterns with the relatively uniform European strains.     

The current MLVA typing scheme for <Emphasis Type="Italic">Enterococcus faecium</Emphasis> is less discriminatory than MLST and PFGE for epidemic-virulent,hospital-adapted clonal types

Background  

MLVA (multiple-locus variable-number tandem repeat analysis) is a reliable typing technique introduced recently to differentiate also isolates of Enterococcus faecium. We used the established VNTR (variable number of tandem repeats) scheme to test its suitability to differentiate 58 E. faecium isolates representing mainly outbreaks and clusters of infections and colonizations among patients from 31 German hospitals. All isolates were vancomycin-resistant (vanA type). Typing results for MLVA are compared with results of macrorestriction analysis in PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing).     

Pulsed-field gel electrophoresis fingerprinting for identification of Azospirillum species

Pulsed-field gel electrophoresis (PFGE) was used to obtain macrorestriction fingerprints of restriction enzyme-cut DNA of natural isolates of Azospirillum spp. Metabolic profiles, along with other phenotypic characteristics, were compared with these fingerprints to differentiate among the azospirilla isolates. A wide diversity of phenotypes (e.g., colony color, motility, and accumulation of poly--hydroxybutyrate granules) was observed among the natural isolates of azospirilla. PFGE revealed that TCTAGA, the sequence recognized by Xba1, is rare in the genome of azospirilla. The PFGE fingerprint revealed that azospirilla associated with different crops have a very similar genetic background. PFGE fingerprints were more consistent in the identification of azospirilla isolates from specific hosts than the metabolic fingerprints. For further differentiation at strain level, metabolic, physiological, and morphological profiles provide additional information.Journal No. J-2920 of the Montana Agricultural Experiment Station.     

Development of a Pulsed-Field Gel Electrophoresis (PFGE) method for molecular typing of clinical isolates of Arcanobacterium haemolyticum

We developed a two-block PFGE method to study molecular variation among clinical isolates of Arcanobacterium haemolyticum, an often overlooked human pathogen. Three main macrorestriction profiles were defined among 15 isolates. PFGE was an objective method for characterizing A. haemolyticum and may be useful in molecular epidemiological studies of this organism.     

Use of pulsed-field gel electrophoresis and flagellin gene typing in identifying clonal groups of Campylobacter jejuni and Campylobacter coli in farm and clinical environments

Although campylobacters have been isolated from a wide range of animal hosts, the association between campylobacters isolated from humans and animals in the farm environment is unclear. We used flagellin gene typing and pulsed-field gel electrophoresis (PFGE) to investigate the genetic diversity among isolates from animals (cattle, sheep, and turkey) in farm environments and sporadic cases of campylobacteriosis in the same geographical area. Forty-eight combined fla types were seen among the 315 Campylobacter isolates studied. Six were found in isolates from all four hosts and represented 50% of the total number of isolates. Seventy-one different SmaI PFGE macrorestriction profiles (mrps) were observed, with 86% of isolates assigned to one of 29 different mrps. Fifty-seven isolates from diverse hosts, times, and sources had an identical SmaI mrp and combined fla type. Conversely, a number of genotypes were unique to a particular host. We provide molecular evidence which suggests a link between campylobacters in the farm environment with those causing disease in the community.     

Use of Pulsed-Field Gel Electrophoresis and Flagellin Gene Typing in Identifying Clonal Groups of Campylobacter jejuni and Campylobacter coli in Farm and Clinical Environments

Although campylobacters have been isolated from a wide range of animal hosts, the association between campylobacters isolated from humans and animals in the farm environment is unclear. We used flagellin gene typing and pulsed-field gel electrophoresis (PFGE) to investigate the genetic diversity among isolates from animals (cattle, sheep, and turkey) in farm environments and sporadic cases of campylobacteriosis in the same geographical area. Forty-eight combined fla types were seen among the 315 Campylobacter isolates studied. Six were found in isolates from all four hosts and represented 50% of the total number of isolates. Seventy-one different SmaI PFGE macrorestriction profiles (mrps) were observed, with 86% of isolates assigned to one of 29 different mrps. Fifty-seven isolates from diverse hosts, times, and sources had an identical SmaI mrp and combined fla type. Conversely, a number of genotypes were unique to a particular host. We provide molecular evidence which suggests a link between campylobacters in the farm environment with those causing disease in the community.     

Molecular diversity of Photobacterium damselae ssp. piscicida from cultured amberjacks (Seriola spp.) in Japan by pulsed-field gel electrophoresis and plasmid profiles

AIMS: This study deals with a pulsed-field gel electrophoresis (PFGE) for discriminating between the genetic variants of Photobacterium damselae ssp. piscicida, and characterizing of Japanese field isolates by PFGE together with plasmid profiles and antimicrobial resistances. METHODS AND RESULTS: A total of 74 field isolates from cultured Japanese amberjacks were used for PFGE. SmaI and NotI enabled to clearly differentiate strains and we obtained 24 of combined PFGE profiles which were distinct from those of classical Japanese and USA reference strains, and classified them into three groups (Ia-Ic). By plasmid size, we could classify these field isolates into three plasmid types, pA-pC. The predominant PFGE-type Ia was closely associated with plasmid-type pA, and Ib showed a moderate association with pB. Ic was closely associated with pC, and multiresistant isolates were not observed in this type. Whole-genomic variations were also observed between isolates having identical detection areas, fish species and detection-date by PFGE. CONCLUSION: Molecular diversity of P. damselae ssp. piscicida could be detected by PFGE, and some relations among the PFGE-type, plasmid-type and antimicrobial resistances were observed in Japanese field isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that some genetic transition might have occurred in P. damselae ssp. piscicida around the Japanese seas, and PFGE can be a valuable tool for the epidemiological study of this highly homogeneous subspecies.     

Comparison of ribotyping,randomly amplified polymorphic DNA,and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis

Brown ring disease, caused by Vibrio tapetis, is an important pathological problem in different species of cultured clams. In order to evaluate the genetic diversity of the pathogen, twenty-seven isolates of V tapetis with different origin were screened by ribotyping (RT), pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA PCR (RAPD). Restriction with PvuII, SalI, and SmaI gave 2 RT patterns, differentiating in all cases the strain 0202RD isolated from carpet-shell clams (Ruditapes decussatus) from the other strains tested. The use of NotI generated strain specific PFGE profiles, which could be grouped in two main clusters. Cluster 1 grouped all but one strain and was subdivided into six PFGE subtypes (1a to 1f) which joined at a similarity level of 75.6%. Cluster 2 included again only the isolate 0202RD. RAPD analysis yielded the same results with three different primers, this method being able to differentiate the isolates from R. decussatus from those isolated from other clam species. Of the three techniques evaluated, PFGE was the most discriminating of the three techniques evaluated, followed in discriminating power by RAPD and RT tests. On the basis of the results obtained, we conclude that the RAPD procedure, which is more rapid and easier to perform than the other techniques, shows to be very useful to analyze large amounts of strain collections from an epidemiological monitoring stanpoint. In addition, PFGE is of great utility to evaluate the genetic diversity of strains involved in an outbreak and to study the spreading of a specific clone.     

Genomic Diversity of Field Isolates of Burkholderia solanacearum in Japan

Genomic differences between 18 isolates of Burkholderia solanacearum originated from diseased plants cultivated in nine different agricultural fields were examined by using four DNA-based methods: repetitive DNA polymorphism analysis, restriction-site analysis of polymerase chain reaction products, random amplified polymorphic DNA (RAPD) analysis, and macrorestriction analysis. Genomic diversity among the isolates was detected by RAPD and macrorestriction analysis. The latter revealed the regional variation of the genome structure among the isolates, suggesting that B. solanacearum populations consist of a number of independent clonal lines.     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

Use of RAPD and AFLP markers to identify inter- and intraspecific hybrids of Mentha

Three controlled crosses were carried out involving Mentha arvensis and Mentha spicata [M. spicata CIMAP/C30 x M. spicata CIMAP/C33 (cv. Neera); M. arvensis CIMAP/C18 x CIMAP/C17 (cv. Kalka); and M. arvensis CIMAP/C17 x M. spicata CIMAP/C33]. The parents were subjected to random amplified polymorphic DNA (RAPD) analysis with 80 primers, and polymorphic primers were tested for detecting coinherited RAPD profiles among the progeny of these crosses. Of 50 seedlings tested from each intraspecific cross, all demonstrated dominant profiles with the selected RAPD primers except the detected hybrid from respective crosses. Coinherited markers could be detected with the primers OPJ 01, MAP 06, OPT 08, and OPO 20 for M. arvensis; OPJ 05, OPJ 14, OPO 19, and OPT 09 for M. spicata; and OPJ 07, OPJ 10, OPJ 11, OPJ 14, and OPO 02 for the cross M. arvensis x M. spicata. In our amplified fragment length polymorphism (AFLP) analysis, 40 coinherited marker fragments were identified for the cross involving M. arvensis, 32 for the cross involving M. spicata, and 41 for the interspecific cross between M. arvensis and M. spicata. In all crosses, similarity values between the parents were less than those between the parents and the hybrids. Although RAPD markers are generally considered dominant, it is possible to identify a few codominant markers that behave like restriction fragment length polymorphism (RFLP) markers. This molecular marker system may be helpful in rapidly screening out hybrids in crops where cross-pollination is a problem.     

Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analyses.

The intraspecific genetic diversity of Oenococcus oeni, the key organism in the malolactic fermentation of wine, has been evaluated by random amplified polymorphic DNA (RAPD), ribotyping, small-plasmid content, and sequencing of RAPD markers with widespread distribution among the strains. Collection strains representing the diversity of this species have been studied together with some new isolates, many of which were obtained from wines produced by spontaneous malolactic fermentation. The RAPD profiles were strain specific and discerned two main groups of strains coincident with clusters obtained by macrorestriction typing in a previous work. Ribotyping and the conservation of RAPD markers indicates that O. oeni is a relatively homogeneous species. Furthermore, identical DNA sequences of some RAPD markers among strains representative of the most divergent RAPD clusters indicates that O. oeni is indeed a phylogenetically tight group, probably corresponding to a single clone, or clonal line of descent, specialized to grow in the wine environment and universally spread.     

Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

TRACKING DISPERSAL ROUTES: PHYLOGEOGRAPHY OF THE ARCTIC-ANTARCTIC DISJUNCT SEAWEED ACROSIPHONIA ARCTA (CHLOROPHYTA)1

Phylogenetic relationships in the Arctic-Antarctic disjunct seaweed species Acrosiphonia arcta (Dillwyn) J. G. Agardh (Acrosiphoniales, Chlorophyta) were examined using restriction fragment-length polymorphism analysis of the fast-evolving nuclear ribosomal intergenic spacer (IGS) region and random amplified polymorphic DNA (RAPD) markers. Twenty-two isolates collected from 10 different locations in both hemispheres were compared. Five IGS length classes were identified among the 10 locations. Throughout the North Atlantic, IGS regions were found to be extremely homogeneous whereas RAPD patterns revealed subdivided populations that suggest founder effects. Acrosiphonia arcta populations found in the Arctic and North Atlantic oceans are hypothesized to be of Pacific origin. Extensive differences found between Arctic Greenland populations and those in the North Atlantic suggest that colonization of Arctic Greenland occurred as an independent event. Recolonization of the Antarctic peninsula from Southern Chile is favored, whereas the directionality of transequatorial passage along the western coast of the Americas could be in either direction.     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.