Epidemiological typing of Bacillus cereus by amplified fragment length polymorphism

A simple amplified fragment length polymorphism method was developed for the epidemiological typing of Bacillus cereus. The method was applied to 21 cultures from seven food poisoning and eight non-food poisoning incidents. Results were compared with those obtained by conventional serotyping using flagellar antigens and assessed in relation to epidemiological data. Amplified fragment length polymorphism was found to be highly reproducible and 16 different profiles (each unique to the 15 incidents) were recognized. The method was also able to discriminate three subtypes within serotype H1, which is responsible for the majority of the emetic type of B. cereus food poisoning in England and Wales.     

Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.

Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.     

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.     

Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

AIMS: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. METHODS AND RESULTS: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and North American origin from human infections, chickens, turkeys, ducks, sheep and poultry abbatoir effluent were studied by use of a protocol that involved stringent PCR amplification of fragments derived from digestion of genomic DNA with restriction enzymes BglII and Csp6I. The mean similarity value of duplicate profiles of 10 isolates was 91.15%, indicating the method to be reproducible. Numerical analysis of all 73 isolates distinguished 51 subtypes at the 91% similarity level, of which 39 comprised single strains. The remaining 34 isolates were distributed among 12 subtypes, each of which contained strains homogeneous with respect to their respective source of isolation. However, contemporaneous strains from the same source could also be distinguished. CONCLUSIONS: AFLP profiling is an effective method for typing the genetically diverse organism A. butzleri. SIGNIFICANCE AND IMPACT OF THE STUDY: The study represents a comprehensive analysis of the genetic diversity of A. butzleri by use of isolates from six countries spanning three continents and also shows that several distinct A. butzleri genotypes may be found in a given environment. AFLP profiling appears to have considerable potential for molecular epidemiological studies of this ubiquitous emerging pathogen that is implicated as a causative agent of both human and animal disease.     

Estimating nucleotide diversity from random amplified polymorphic DNA and amplified fragment length polymorphism data

A way to estimate the index of nucleotide diversity (pi) from band match frequencies in random amplified polymorphic DNA and amplified fragment length polymorphism data is described. pi is shown to be a simple function of the proportion of mismatched bands between two individuals drawn at random from a population (phi) and the number of discriminating sites in the amplification system. The method is computationally and conceptually simple and avoids some of the assumptions inherent in other approaches: the relationship is independent of the base composition of the target DNA and avoids the bias inherent in estimations of allelic frequencies in dominant systems. Only two individuals from a population are needed to estimate pi. This economy of material suggests utility of this approach in conservation genetics or other fields where obtaining large samples is impractical or undesirable.     

Molecular characterization of Korean Bacillus anthracis isolates by amplified fragment length polymorphism analysis and multilocus variable-number tandem repeat analysis

We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.     

Genetic diversity of Echinacea species based upon amplified fragment length polymorphism markers.

The taxonomy of Echinacea is based on morphological characters and has varied depending on the monographer. The genus consists of either nine species and four varieties or four species and eight varieties. We have used amplified fragment length polymorphisms (AFLP) to assess genetic diversity and phenetic relationships among nine species and three varieties of Echinacea (sensu McGregor). A total of 1086 fragments, of which approximately 90% were polymorphic among Echinacea taxa, were generated from six primer combinations. Nei and Li's genetic distance coefficient and the neighbor-joining algorithm were employed to construct a phenetic tree. Genetic distance results indicate that all Echinacea species are closely related, and the average pairwise distance between populations was approximately three times the intrapopulation distances. The topology of the neighbor-joining tree strongly supports two major clades, one containing Echinacea purpurea, Echinacea sanguinea, and Echinacea simulata and the other containing the remainder of the Echinacea taxa (sensu McGregor). The species composition within the clades differs between our AFLP data and the morphometric treatment offered by Binns and colleagues. We also discuss the suitability of AFLP in determining phylogenetic relationships.     

Population structure of Salmonella investigated by amplified fragment length polymorphism

AIMS: This study was undertaken to investigate the usefulness of amplified fragment length polymorphism (AFLP) in determining the population structure of Salmonella. METHODS AND RESULTS: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination that is novel in Salmonella. Both species S. bongori and S. enterica and all subsp. of S. enterica were represented with emphasis on S. enterica subsp. enterica using a local strain collection and strains from the Salmonella Reference Collection B (SARB). The amplified fragments were used in a band-based cluster analysis. The tree resulting from the subgroup analysis clearly separated all subgroups with high bootstrap values with the species S. bongori being the most distantly related of the subgroups. The tree resulting from the analysis of the SARB collection showed that some serotypes are very clonal whereas others are highly divergent. CONCLUSIONS: AFLP clearly clustered strains representing the subgroups of Salmonella together with high bootstrap values and the serotypes of subspecies enterica were divided into polyphyletic or monophyletic types corresponding well with multilocus enzyme electrophoresis (MLEE) and sequence-based studies of the population structure in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP with the enzyme combination BglII and BspDI allows discrimination of individual strains and provides evidence for the usefulness of AFLP in studies of population structure in Salmonella.     

Genetic diversity assessed by amplified fragment length polymorphism analysis of the parasitic nematode Dictyocaulus viviparus the lungworm of cattle

We have examined the population genetic structure in a collection of nine isolates of the parasitic lungworm Dictyocaulus viviparus. Eight of the isolates were sampled from cattle in geographically separated farms throughout south-central Sweden, and one isolate was a laboratory strain that has been maintained in experimentally infected calves for almost four decades. A total of 72 worms were examined, with eight individual worms from the same individual host representing each isolate. The genetic variation as revealed by amplified fragment length polymorphism analysis using four selective primer combinations was high. Depending on the primer combination a total of 66-79 restriction fragments were amplified, with 26-44 peaks of similar complexity from each of the isolates. The heterozygosity within populations was relatively small, as were the population mutation and immigration rates, which seemed to be in neutral equilibrium. The genetic diversity was therefore reasonably well structured in the field; and the laboratory isolate was quite distinct from the field samples. There was no relationship between the patterns of genetic diversity and the geographical proximity of the farms. The estimates of heterozygosity were much larger and more consistent than those previously estimated for this nematode species using mitochondrial sequencing, and the genetic structuring was thus much less pronounced and the gene flow greater. We attribute these differences in estimation to the broader sampling of loci available using amplified fragment length polymorphism markers, which may therefore constitute a superior technique for the study of patterns of lungworm diversity. Furthermore, the data estimating gene flow for D. viviparus was less than previously reported for closely related species in North America. This might be related to different rates of movements of infected hosts. It seems likely that lungworm infections are rather persistent on different farms, and the sudden outbreaks of disease that can be observed with host movements are most likely to be related to the introduction of susceptible stock.     

Statistical analysis of amplified fragment length polymorphism data: a toolbox for molecular ecologists and evolutionists

Recently, the amplified fragment length polymorphism (AFLP) technique has gained a lot of popularity, and is now frequently applied to a wide variety of organisms. Technical specificities of the AFLP procedure have been well documented over the years, but there is on the contrary little or scattered information about the statistical analysis of AFLPs. In this review, we describe the various methods available to handle AFLP data, focusing on four research topics at the population or individual level of analysis: (i) assessment of genetic diversity; (ii) identification of population structure; (iii) identification of hybrid individuals; and (iv) detection of markers associated with phenotypes. Two kinds of analysis methods can be distinguished, depending on whether they are based on the direct study of band presences or absences in AFLP profiles ('band-based' methods), or on allelic frequencies estimated at each locus from these profiles ('allele frequency-based' methods). We investigate the characteristics and limitations of these statistical tools; finally, we appeal for a wider adoption of methodologies borrowed from other research fields, like for example those especially designed to deal with binary data.     

Restriction fragment length polymorphism diversity in soybean

Summary Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.     

Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism

AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown. Previous attempts to fingerprint E. amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium. Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen. METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension. PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains. Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns. CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E. amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen. The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination. This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E. amylovora strains.     

Amplification of reproducible allele markers for amplified fragment length polymorphism analysis.

A procedure for amplification by PCR of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis is presented. We have prepared markers for the allelic products of the VNTR loci D1S80 (MCT118) and D17S30 (YNZ22) and for the hypervariable VNTR locus close to the 3' end of the apolipoprotein B gene (apoB) by re-amplifying a mixture of PCR products from individuals with known alleles. These allele markers allow precise and discrete determination of the VNTR alleles at these loci using the Amp-FLP technique that should prove suitable in forensic analyses, paternity testing and population studies.     

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Genetic diversity of clinical and environmental isolates of Vibrio cholerae determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.     

An amplified fragment length polymorphism map of the silkworm

The silkworm (Bombyx mori L.) is a lepidopteran insect with a long history of significant agricultural value. We have constructed the first amplified fragment length polymorphism (AFLP) genetic linkage map of the silkworm B. mori at a LOD score of 2.5. The mapping AFLP markers were genotyped in 47 progeny from a backcross population of the cross no. 782 x od100. A total of 1248 (60.7%) polymorphic AFLP markers were detected with 35 PstI/TaqI primer combinations. Each of the primer combinations generated an average of 35.7 polymorphic AFLP markers. A total of 545 (44%) polymorphic markers are consistent with the expected segregation ratio of 1:1 at the significance level of P = 0.05. Of the 545 polymorphic markers, 356 were assigned to 30 linkage groups. The number of markers on linkage groups ranged from 4 to 36. There were 21 major linkage groups with 7-36 markers and 9 relatively small linkage groups with 4-6 markers. The 30 linkage groups varied in length from 37.4 to 691.0 cM. The total length of this AFLP linkage map was 6512 cM. Genetic distances between two neighboring markers on the same linkage group ranged from 0.2 to 47 cM with an average of 18.2 cM. The sex-linked gene od was located between the markers P1T3B40 and P3T3B27 at the end of group 3, indicating that AFLP linkage group 3 was the Z (sex) chromosome. This work provides an essential basic map for constructing a denser linkage map and for mapping genes underlying agronomically important traits in the silkworm B. mori L.     

Olive genetic diversity assessed using amplified fragment length polymorphisms

 Amplified fragment length polymorphism (AFLP) analysis was used to study the genetic variation within and among populations of genus Olea. A group of genotypes, all of them cultivated varieties of a single species, Olea europaea, was compared with wild olives and with a group of individuals belonging to different Olea species. Five primer combinations were used which produced about 290 polymorphic bands. The data obtained were elaborated with the Nei’s genetic similarity coefficient, applying different clustering methods and the Principal Coordinate Analysis. Cultivars, wild olives and North-West African species formed groups clustering together at a similarity level of 0.56, while the Olea species from East Africa and Asia grouped separately. Species from the Indian Ocean and Australia showed the highest diversity. We hypothesize that cultivars and wild plants are different forms of the same O. europaea species. The Olea from East Africa and Asia may be assigned to a different species, while the role of O. laperrini as well as that of O. maroccana as an intermediary form is confirmed. Received: 30 April 1998 / Accepted: 13 August 1998     

Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis

Amplified fragment length polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos et al. 1995). The method simply surveys the genome for length and sequence polymorphisms. The AFLP pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. We are using AFLP analysis to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of Bacillus anthracis strains shows very little variability among different isolates (Keim et al. 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information cannot be rapidly generated by other means. AFLP sample analysis quickly generates a very large amount of molecular information about microbial genomes. However, this information cannot be analysed rapidly using manual methods. We are developing a large archive of electronic AFLP signatures that is being used to identify isolates collected from medical, veterinary, forensic and environmental samples. We are also developing the computational packages necessary to rapidly and unambiguously analyse the AFLP profiles and conduct a phylogenetic comparison of these data relative to information already in our database. We will use this archive and the associated algorithms to determine the species identity of previously uncharacterized isolates and place them phylogenetically relative to other microbes based on their AFLP signatures. This study provides significant new information about microbes with environmental, veterinary and medical significance. This information can be used in further studies to understand the relationships among these species and the factors that distinguish them from one another. It should also allow the identification of unique factors that contribute to important microbial traits, including pathogenicity and virulence. We are also using AFLP data to identify, isolate and sequence DNA fragments that are unique to particular microbial species and strains. The fragment patterns and sequence information provide insights into the complexity and organization of bacterial genomes relative to one another. They also provide the information necessary for the development of species-specific polymerase chain reaction primers that can be used to interrogate complex samples for the presence of B. anthracis, other microbial pathogens or their remnants.     

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.