Genotyping of thermotolerant Campylobacter from poultry slaughterhouse by amplified fragment length polymorphism

AIM: To examine the occurrence, diversity and transmission of Campylobacter in a poultry slaughterhouse. METHODS AND RESULTS: During a 4-week period, a slaughterhouse was sampled alternately during slaughtering and the following mornings post-disinfection. Samples were taken from poultry at six stages in the slaughter process and from 25 environmental sites. For positive broiler flocks slaughtered on one occasion, 92% and 48% of the environmental sites were positive during slaughter and post-disinfection, respectively. For positive laying hen flocks slaughtered on three occasions, 8-56% and 12-20% of the environmental sites were positive during slaughter and post-disinfection, respectively. Genetic fingerprinting by amplified fragment length polymorphism (AFLP) of the 109 isolates obtained resulted in 28 different AFLP clones. Five AFLP clones were present for more than 1 week. CONCLUSIONS: Slaughtering of Campylobacter-positive broilers resulted in extensive contamination of the slaughterhouse, including the air. A high proportion of the laying hen flocks were Campylobacter positive, but these caused less environmental contamination than the broilers. This, together with the freezing of all layer carcasses, results in a lower public health risk from laying hens, when compared with broilers. Significance and Impact of the Study: When slaughtering Campylobacter-positive broilers, the implementation of preventive measures is important to reduce contamination of negative carcasses and to protect the workers against infection.     

放线菌的单酶切AFLP基因分型研究

使用MseI限制性内切酶对放线菌链霉菌属中的12株菌基因组DNA进行酶切,与接头连接后,引物使用一个选择性碱基对模板进行PCR扩增,PCR产物在琼脂糖凝胶上进行电泳检测。结果表明,对于MseI内切酶产生的模板,选择性碱基采用A、T、C和G都能够获得清晰丰富的条带。MseI内切酶产生的图谱上有72个位点,多态性位点71个,达98．6％。因此,使用MseI内切酶适合构建放线菌的单酶切AFLP指纹图谱。     

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

AFLPMax: a user-friendly application for computing the optimal number of amplified fragment length polymorphism markers needed in phylogenetic reconstruction

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic inference especially in non-model species. Frequently, trees obtained with other nuclear or mitochondrial markers or with morphological information need additional resolution, increased branch support, or independent data sources (i.e. unlinked loci). In such cases, the use of AFLPs is a quick and cheap option. Computer simulation has shown that dominant AFLP markers lead to less accurate tree topologies than bi-allelic codominant markers such as SNPs, but this difference becomes negligible for shallow trees when using AFLP data sets that include a sufficiently large number of characters. Thus, determining how many AFLP characters are required to recover a given phylogeny is a key issue regarding the appropriateness of AFLPs for phylogenetic reconstruction. Here, we present a user-friendly, java-based graphical interface, AFLPMax, which executes an automatic pipeline of different programs providing the user with the optimal number of AFLP characters needed to recover a given phylogeny with high accuracy and support. Executables for Windows, linux and MacOS X operating systems, source code and user manual are available from: http://webs.uvigo.es/acraaj/AFLPMax.htm.     

Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles

AIMS: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. METHODS AND RESULTS: Field strains (n = 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters. CONCLUSIONS: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic relatedness among all 'Penner' serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.     

In vitro propagation, restriction fragment length polymorphism, and random amplified polymorphic DNA analyses of Angelica plants

Angelica acutiloba, a medicinal plant used as a natural medicine Touki, was clonally propagated through axillary buds in vitro. No substantial differences were found in the random amplified polymorphic DNA (RAPD) pattern between the original A. acutiloba and the plant propagated in vitro, suggesting no changes in the DNA sequences and structure during in vitro propagation. The genetic similarities of several Angelica plants were investigated by restriction fragment length polymorphism (RFLP) and RAPD analyses. The RFLP and RAPD patterns of A. sinensis Diels were substantially different from those of A. acutiloba. Using ten different restriction enzymes, no RFLP was observed in the varieties of A. acutiloba. By RAPD analysis, A. acutiloba varieties can be classified into two major subgroups, i.e., A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyamae Hikino. The varieties of A. acutiloba Kitagawa in Japan and Angelica spp. in northeast China exhibited a very close genetic relationship. Received: 13 March 1998 / Revision received: 28 July 1998 / Accepted: 21 August 1998     

Characterisation of persistent and sporadic Listeria monocytogenes strains by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP)

This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants. In each plant persistent and sporadic strains were selected on the basis of PFGE typing results. A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study. PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information. The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively. The combination of PFGE and AFLP typing results yielded a total of 48 genotypes. Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only. Our results showed that L. monocytogenes strains causing persistent contamination differ from sporadic strains. In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups. These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.     

Genetic stability of strains preserved on LENTICULE discs and by freeze-drying: a comparison using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis, a high-resolution genome fingerprinting method, was used to ascertain the DNA integrity of bacterial strains during preservation by lenticulation and by traditional freeze-drying into glass ampoules. This was achieved by comparing FAFLP genotypes of a range of paired bacterial isolates recovered from LENTICULE discs (preserved between 1995 and 2004) and from freeze-dried (FD) cultures in glass ampoules (preserved between 1966 and 2000). A choice of two endonuclease combinations EcoRI/MseI or HindIII/HhaI was used for FAFLP analysis of the five different bacterial genera comprising of 10 strains. Each of these 10 strains exhibited unique FAFLP profiles. However, there were no detectable differences between the FAFLP profiles for each of the individual strains, irrespective of their preservation format or their year of preservation. Thus, the FAFLP data suggests that LENTICULE production does not result in any detectable genetic changes during drying onto LENTICULE discs and storage for at least 5 years. The provision of such FD reference cultures on LENTICULE discs rather than FD glass ampoules will provide a cost-effective format that is easier to use.     

Complete exclusion of nonsires in an analysis of paternity in a natural plant population using amplified fragment length polymorphism (AFLP)

The utility of the new polymerase chain reaction (PCR)-based multilocus DNA fingerprinting technique amplified fragment length polymorphism (AFLP) for paternity analysis in natural plant populations was assessed. In a natural population of 25 plants of Persoonia mollis (Proteaceae), three AFLP primer pairs generated 147 dominant loci. Of these, 125 (85%) were polymorphic, with a mean recessive allele frequency of 0.735. The theoretical expected percentage of offspring for which all males except the true father can be excluded ( P _ET) was 99.9% for this population. The estimates of P _ET drop marginally to 99.6% and 97.6% for larger populations of 100 and 1000 individuals, respectively. A preliminary investigation confirmed the power of AFLP for paternity analysis by assigning paternity, or excluding all known potential sires, for 242 of 252 (96.0%) naturally pollinated seeds. Ambiguous paternity for the remaining 10 seeds was quickly resolved by utilizing two further AFLP primer pairs, ultimately generating over 200 polymorphic loci and resulting in the exclusion of all nonsires for all 252 (100%) seeds. This study highlights the utility of AFLP for paternity analysis because: (i) it generates sufficiently large numbers of highly reproducible polymorphic loci, that are (ii) quickly and accurately scored using an automated DNA sequencer and dedicated software, and (iii) unlike microsatellites, requires no sequence knowledge so it is more easily applied to new study species.     

Amplified fragment length polymorphism (AFLP) analysis of genetic variation in Moringa oleifera Lam

Moringa oleifera is an important multipurpose tree introduced to Africa from India at the turn of this century. Despite limited knowledge of the levels of genetic diversity and relatedness of introduced populations, their utilization as a source of seed for planting is widespread. In order to facilitate reasoned scientific decisions on its management and conservation and prepare for a selective breeding programme, genetic analysis of seven populations was performed using amplified fragment length polymorphism (AFLP) markers. The four pairs of AFLP primers ( Pst I/ Mse I) generated a total of 236 amplification products of which 157 (66.5%) were polymorphic between or within populations. Analysis of molecular variance ( AMOVA ) revealed significant differences between regions and populations, even though outcrossing perennial plants are expected to maintain most variation within populations. A phenetic tree illustrating relationships between populations suggested at least two sources of germplasm introductions to Kenya. The high levels of population differentiation detected suggest that provenance source is an important factor in the conservation and exploitation of M. oleifera genetic resources.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.     

Amplified fragment length polymorphism (AFLP) analysis of the genetic structure of the zebra mussel, Dreissena polymorpha, in the Mississippi River

1. We predicted that zebra mussel, Dreissena polymorpha (Pallas), genetic structure in the Mississippi River would follow a model of invasive species genetics, which predicts low genetic structure among populations of recently established species. This prediction was upheld in our previous genetic study using allozymes, however, one locus yielded anomalous results. 2. We employed amplified fragment length polymorphism (AFLP) analysis as a neutral marker to assess the amount of genetic structure within and among populations, and as a test of expected population structure from both invasion genetic theory, and the results from our previous study. 3. There was greater spatial differentiation, as measured by F_st, observed using AFLP's than for allozymes (P < 0.001). There was no evidence that AFLP variation conformed to an isolation by distance model, and genetic relationships of populations, as measured by AFLP markers, were not similar to those detected in our allozyme survey. 4. The lack of concordance between these two genetic marker systems probably reflects their differential responses to drift, migration, and selection occurring during this rapid invasion. Strong population structure is counter to predictions that populations of invasive species will not be differentiated, as with observations based on allozyme markers. Therefore, newly established species may require genetic surveys using multiple marker systems to evaluate population structure.     

Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes

Summary. A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were Taq I and Msp I. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.     

Geographic pattern of genetic variation in the European globeflower Trollius europaeus L. (Ranunculaceae) inferred from amplified fragment length polymorphism markers

The distribution of genetic variation and the phylogenetic relationships between 18 populations of the arctic-alpine plant Trollius europaeus were analysed in three main regions (Alps, Pyrenees and Fennoscandia) by using dominant AFLP markers. Analysis of molecular variance revealed that most of the genetic variability was found within populations (64%), although variation among regions (17%) and among populations within regions (19%) was highly significant (P < 0.001). Accordingly, the global fixation index FST averaged over loci was high (0.39). The among-population differentiation indicates restricted gene flow, congruent with limited dispersal of specific globeflower's pollinating flies (Chiastocheta spp.). Within-population diversity levels were significantly higher in the Alps (mean Nei's expected heterozygosity HE = 0.229) than in the Pyrenees (HE= 0.197) or in Fennoscandia (HE = 0.158). This finding is congruent with the species-richness of the associated flies, which is maximum in the Alps. We discuss the processes involved in shaping observed patterns of genetic diversity within and among T. europaeus populations. Genetic drift is the major factor acting on the small Pyrenean populations at the southern edge of T. europaeus distribution, while large Fennoscandian populations result probably from a founder effect followed by demographic expansion. The Alpine populations represent moderately fragmented relics of large southern ancestral populations. The patterns of genetic variability observed in the host plant support the hypothesis of sympatric speciation in associated flies, rather than recurrent allopatric speciations.     

Evaluation of genetic diversity and germ plasm identification of 44 species, clones, and cultivars from 5 sections of the genusPopulus based on amplified fragment length polymorphism analysis

The genusPopulus L. (Salicaceae) can be divided into 5 sections with distribution throughout the world. Accurate identification ofPopulus clones and species is essential for effective selection, breeding, and management of genetic resources. In this study, amplified fragment length polymorphism (AFLP) analysis, which was reported as a reliable technique with high efficiency in detecting polymorphism, was used to conduct analyses of genetic diversity and variety identification of 44 species, clones, and cultivars ofPopulus that represent a wide range of breeding and commercially available germplasms. Cluster analysis of the 44 samples was carried out, and a dendrogram of genetic relatedness was developed on the basis of the AFLP data. DNA fingerprints of the 44 samples were developed from 12 selected bands amplified with 2 primer combinations (M-CAG/E-TA and M-CAG/E-TC). Each sample has its unique fingerprint pattern and can be distinguished from the others. Furthermore, 1 specific AFLP band of the cultivarPopulus canadensis cl. Guariento coming from fragments amplified by primer combination M-CTC/E-AG was successfully converted into a sequence-characterized amplified region (SCAR) marker. The results indicate that AFLP analysis should be considered as the preferred technique for the study of polymorphism inPopulus. This research is the first report concerning the use of AFLP analysis in genetic diversity and germplasm identification among all sections ofPopulus.     

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.     

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated from unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coli var. hyoilei strains, were identified. These relationships corresponded to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp.     

Comparative analysis of amplified fragment length polymorphism and pulsed field gel electrophoresis in a hospital outbreak and subsequent endemicity of ampicillin-resistant Enterococcus faecium

Reliable molecular methods for determination of relatedness between bacterial isolates have become increasingly important to evaluate outbreaks and endemic situations with nosocomial pathogens. In the present study Simpson's index of diversity with calculated confidence intervals was used to compare amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) analysis of a hospital outbreak of ampicillin-resistant Enterococcus faecium and subsequent endemicity. The outbreak, in a Norwegian tertiary hospital, of infections caused by these enterococci started in 1995 and increased in 1996 after which the situation turned endemic. The purpose of this study was to compare the two methods in this setting and to determine the length of time during an outbreak that these methods are sufficiently valid to be of value for hospital infection control efforts. One hundred and sixty clinical isolates from urine specimens collected during the period 1995-1999 were included. The findings indicate that PFGE and AFLP are equally discriminative and could in this setting be used for typing purposes over the whole 5-year period.