Isoenzyme analysis of Babesia microti infections in humans

We used high-resolution polyacrylamide gradient gel electrophoresis (PGGE) to separate four babesial enzymes to aid in the identification of two Babesia microti isolates established in hamsters. The isolates were compared to two different hamsters passages of the "Gray" strain. All isoenzymes patterns from the two isolates and the "Gray" strain were similar except glucose phosphate isomerase (GPI) from one of the "Gray" strain passages. It showed a polymorphic GPI pattern as opposed to a monomorphic GPI pattern seen in the other "Gray" strain passage and the two isolates. The observed differences suggested that some population of B. microti are capable of having polymorphic GPI, that the "Gray" strain originally contained (and may still contain) a heterogeneous population of B. microti, and that the population possessing polymorphic GPI was selected over that with monomorphic GPI. This information was obtained by a PGGE method that eliminated hemoglobin from gels and allowed, for the first time, detection of babesial leucine amino peptidase (LAP) and isocitrate dehydrogenase (IDH). In addition, this method provided molecular weight estimations on babesial GPI, LAP, IDH, and glutamate dehydrogenase (GDH), and it proved useful in the identification and characterization of the B. microti isolates.     

Isozyme Analysis in a Genetic Collection of Amaranths (<Emphasis Type="Italic">Amaranthus</Emphasis> L.)

In various populations of the cultivated and weedy amaranth species, the electrophoretic patterns of alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and malic enzyme (Me) were studied. In total, 52 populations and two varieties (Cherginskii and Valentina) have been examined. Allozyme variation of this material was low. Irrespective of species affiliation, 26 populations and two varieties were monomorphic for five enzymes; a slight polymorphism of three, two, and one enzymes was revealed in three, nine, and fourteen populations, respectively. A single amaranth locus, Adh, with two alleles, Adh F and Adh S, controls amaranth ADH. Two alleles, common Gdh S and rare Gdh F, control GDH; no heterozygotes at this locus were found. The MDH pattern has two, the fast- and slow-migrating, zones of activity (I and II, respectively). Under the given electrophoresis conditions, the fast zone is diffuse, whereas slow zone is controlled by two nonallelic genes, monomorphic Mdh 1 and polymorphic Mdh 2 that includes three alleles: Mdh 2-F, Mdh 2-N, and Mdh 2-S. Low polymorphism of IDH and Me was also found, though their genetic control remains unknown.     

Isozyme analysis in a genetic collection of amaranths (Amaranthus L)

In various populations of the cultivated and weedy amaranth species, the electrophoretic patterns of alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and malic enzyme (Me) were studied. In total, 52 populations and two varieties (Cherginskii and Valentina) have been examined. Allozyme variation of this material was low. Irrespective of species affiliation, 26 populations and two varieties were monomorphic for five enzymes; a slight polymorphism of three, two, and one enzymes was revealed in three, nine, and fourteen populations, respectively. A single amaranth locus, Adh, with two alleles, Adh F and Adh S, controls amaranth ADH. Two alleles, common Gdh S and rare Gdh F, control GDH; no heterozygotes at this locus were found. The MDH pattern has two, the fast- and slow-migrating, zones of activity (I and II, respectively). Under the given electrophoresis conditions, the fast zone is diffuse, whereas slow zone is controlled by two nonallelic genes, monomorphic Mdh 1 and polymorphic Mdh 2 that includes three alleles: Mdh 2-F, Mdh 2-N, and Mdh 2-S. Low polymorphism of IDH and Me was also found, though their genetic control remains unknown.     

Borrelia burgdorferi and Babesia microti: efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)

In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically, spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas.     

Identification of Molecular Marker and Aggressiveness for Different Groups of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates Causing Spot Blotch Disease in Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

One hundred fifty-five isolates of Bipolaris sorokiniana of wheat were studied for their morphopathological characterization. These isolates were grouped in five categories--black, brown/dull black, gray cottony growth, dull white/greenish black, and white--on the basis of their growth pattern. The frequency of the black suppressed type was maximum (45.63%), whereas the white isolate displayed lowest frequency (6.96%) in the natural population. Twenty RAPD (random amplified polymorphic DNA) primers were used to observe the variability among the identified groups of B. sorokininana. From each group, eight random isolates were investigated. A total of 143 bands were amplified, out of which 107 (74.83%) were polymorphic and 36 (25.17%) were monomorphic. On an average, the total numbers of bands generated per primer were 7.15, of which 5.35 and 1.80 were polymorphic and monomorphic, respectively. Dendrograms based on molecular polymorphism unveiled a considerable amount of diversity among the isolates. Specific DNA bands were identified for selected isolates. The distinct markers appeared to be potential enough to be employed as genetic fingerprints for future strain identification and classification. The study indicated that the RAPD primers provide an easy, rapid, and simple technique for the preliminary assessment of genetic diversity among the fungal isolates.     

Some characteristics of genetic variants of Tubifex tubifex (Müller, 1774) (Oligochaeta: Tubificidae) in laboratory cultures

Genetic variants of the oligochaete Tubifex tubifex were identified with enzyme electrophoresis and subsequently reared in laboratory cultures. Three types are abundant in field populations. Individuals that show homozygotic bands of glucosephosphate-isomerase (GPI) 22 together with isocitrate-dehydrogenase (IDH) 35 were labeled Type A. Type B is characterized by GPI 23 together with IDH 11 and Type C is characterized by GPI 11 with either IDH 34 or IDH 33. Initial results on freshweights of adults and cocoon production revealed differences between the two main types, A and B. In the same period, Type B reached higher weights and produced five times as many cocoons as Type A, whereas number of eggs per cocoon were not different between these Types. Type B also had the lowest mortality in 16-week experiments with changing temperatures.     

Isozymatic Differentiation in Local Population of Glycine soja Sieb. & Zucc.

The biochemical genetic structure and variation among local population of Glycine soja Sieb. & Zucc. were investigated based on isozyme analysis using the techniques of polyacrylamide gel electrophoresis. The isoenzyme zymography of 6 enzymes viz malate dehydrogenase (MDH), peroxidase (PER), adenosine triphosphatase (ATPase), amylase (AMY), esterase (EST) and isocitric dehydrogenase (IDH) of 14 culture seedlings were respectively compared. Isozymatic analysis revealed high genetic variation in the population of G. soja. MDH, PER, ATPase, AMY are polymorphic. ATPase has the highest polymorphic index (PI=O. 1582). EST and IDH are monomorphic for all populations. The average population heterozygosity (He) was 0. 3141, and the average genetic distance (Da) among the 14 samples is 0. 1512. Cluster analysis and canonical analysis showed no correlation existed between the population's biochemical genetic structure and its environment. It was concluded that mutation could be the major cause of the high enzymatic polymorphism in population; and the mechanism that keeps the polymorphism could be random drift sampling strategy for conservation of crop genetic resources was also put forward.     

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.     

棉铃虫地理种群的等位酶变异

利用聚丙烯酰胺梯度凝胶电泳检测了棉铃虫Helicoverpa armigera的13种等位酶：α-磷酸甘油脱氢酶(α-GPDH)、酸性磷酸酯酶(ACPH)、碱性磷酸酯酶(ALP)、醛氧化酶(AO)、酯酶(EST)、谷氨酸草酰乙酸转氨酶(GOT)、己糖激酶(HEX)、亮氨酸氨肽酶(LAP)、乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、苹果酸酶(ME)、磷酸葡萄糖变位酶(PGM)和黄嘌呤脱氢酶(XDH),染色采用双染法。对其中9种等位酶的遗传变异进行了分析,包括13个位点,6个位点表现出多态性,7个位点是单态的,其中多态性位点比例为46.15%。AO、GOT、LAP、LDH、ME和XDH计算出棉铃虫的平均杂合度为0.1160,南京、成都、武穴、衡阳和哈密5个种群的平均遗传距离为0.0008～0.0293,平均遗传相似度为0.9707～0.992。棉铃虫种群内存在很高的遗传多态性,而已测定的种群间遗传分化程度较小,种群间没有基因交流的障碍。迁飞阻碍了不同地理种群间的遗传分化。     

Monomorphism of isozyme loci in natural and cultivated amaranth (<Emphasis Type="Italic">Amaranthus</Emphasis> L.) populations

Electrophoretic spectra of alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), and malic enzyme (ME) in different amaranth populations has been studied using a starch gel electrophoresis. 93 populations and 4 cultivars of amaranth have been analyzed. Some populations have been proved to be polymorphic that provided a possibility of a genetic control of the above-mentioned enzymes. The isozyme variability of the studied amaranth populations is low; all studied loci are found to be monomorphic for 73 populations and 4 cultivars. Some populations demonstrate a polymorphism in separate loci (Adh, Mdh 2, Gdh, Idh 1, Idh 2, and Mod 2). The obtained results evidence the presence of a genetic monomorphism in amaranth concerning the loci studied.     

Phenotypic polymorphism and allele differentiation of isozymes in fodder beet,multigerm sugar beet and monogerm sugar beet

Summary Thirteen enzymes (MDH, SDH, LAP, PGM, PX, IDH, GPI, 6PGD, APH, GOT, GDH, ME and SOD) of 3 cultivated beet (B. vulgaris L.) gene pools, comprising 12 accessions of fodder beet, 11 of old multigerm sugar beet and 10 of modern monogerm sugar beet, were investigated using horizontal starch gel electrophoresis. Eleven accessions of primitive or wild B. vulgaris were also included for the comparison of isozymes. Variation in isozyme phenotypes was investigated to detect diversity in the three cultivated forms of beet. Phenotypic variation was observed in all except ME and SOD, which were monomorphic. A high degree of phenotypic polymorphism (Pj) was found in GDH, PGM, IDH, APH and MDH. Differences in phenotypic polymorphism in MDH, GPI and PX were recognized between fodder beet and both sugar beet groups. Average polymorphism for 13 enzymes in both sugar beets was significantly higher than that in fodder beet. For 13 enzymes, the existence of high isozyme diversity in both sugar beet gene pools was revealed. Allele frequencies in 13 alleles of five enzyme-coding loci, Lap, Px-1, Aph-1, Got-2 and Gdh-2, were investigated. New alleles, Px-1 ¹ and Got-2 ¹, were found in fodder beet accessions. No significant differences of average allele frequencies of five loci between fodder beet and both sugar beets were recognized. Several unique alleles and different isozyme phenotypes were observed in the accessions of B. vulgaris ssp. macrocarpa and ssp. adanensis. Future utilization of cultivated beet gene pools for sugar beet breeding is discussed from the viewpoint of genetic resources.     

NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.     

Molecular identification of Babesia parasites isolated from Ixodes ricinus ticks collected in northwestern Poland

In the present study, PCR has been applied to detect and analyze DNA of Babesia spp. extracted from Ixodes ricinus ticks. Collection of I. ricinus was made in 6 forested areas of Zachodniopomorskie Voivodship, Poland, during 2 seasonal peaks of tick activity, i.e., spring and autumn, 2001. In total, 1,328 I. ricinus were collected and processed for PCR with F34 and R323 primers. Babesia spp. was detected in 28 (2% of 1,328 tested) ticks; 26 were identified as B. divergens. The other 2 were identified as B. microti. PCR was conducted with 18S rRNA specific primers and sequencing was processed to precisely identify and compare these isolates with B. microti and B. divergens sequences from Europe, North America, and Asia obtained from the GenBank. Analysis revealed that sequences of B. microti from northwestern Poland are almost identical (99.94%) with those referred to as "Munich strain"; both form a clade different from other European strains, as well as those from Asia and North America (called B. microti, sensu stricto). An investigation performed with B. divergens sequences showed that the sequence from northwestern Poland is 99.94% homologous to an isolate from Ireland ("Purnel"), and differs in just a few nucleotides from other European sequences. Phylogenetic analysis revealed that the sequence of B divergens isolated from Polish ticks form a group that comprise 4 European sequences from Great Britain and Ireland and is, therefore, closely related to other European and North American B. divergnens sequences.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Susceptibility of five strains of mice to Babesia microti of human origin.

One outbred (CF1) and four inbred (BALB/c, C57, CBA and C3H) strains of mice were tested for susceptibility to Babesia microti of human origin. Of these, intact C3H mice developed higher parasitemia than all other intact mice, while BALB/c mice developed the highest parasitemia among splenectomized mice. Susceptibility was not related to H-2 haplotype in any obvious way. Because C3H and BALB/c mice developed relatively high initial peak parasitemias, the parasite was serially passaged in both of these mouse strains in an attempt to increase parasite virulence. After 30 passages in BALB/c and 49 passages in C3H mice over a period of 12 months, maximum parasitemias were 50 times higher than those observed initially. After the peak parasitemias of these two mouse-adapted parasites had stabilized, the relationship between onset and level of maximum parasitemia and number of parasites inoculated was determined. With both C3H- and BALB/c-adapted parasites, as inoculum size increased, the time required to reach maximum parasitemia decreased and the level of maximum parasitemia increased. Studies involving infection of either mouse strain with parasites adapted to the heterologous mouse strain indicated that C3H mice were more susceptible than BALB/c mice to homologous or heterologous parasites. These data suggest that the virulence of B. microti to the mouse can be increased by prolonged passage in this host. Once adaptation to this host species has occurred, virulence appears to be more dependent on the innate susceptibility of the mouse strain than on adaptation of the parasites to a particular strain of mouse.     

Detection of natural foci of babesiosis and granulocytic ehrlichiosis in Russia

The use of microscopy, infection of golden hamsters and the polymerase chain reaction made it possible to find out that about 30% of common red-backed voles (Clethrionomys glareolus), inhabiting the taiga forests of the southern part of the Western Urals (the Chusovskoi district of the Perm region), were infected with Babesia microti and simultaneously (a third of them) with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of granulocytic ehrlichiosis. The sequencing of 18S rDNA of strain "Mys", isolated in Russia, revealed its identity to American B. microti strain GI, pathogenic for humans. The main vector supporting the circulation of B. microti in the natural foci in the region where these investigations were conducted was, seemingly, the tick Ixodes trianguliceps, Thus, for the first time the data proving the presence of reservoir hosts infected with B. microti and granulocytic E. phagocytophila, pathogenic for humans, in Russia were presented.     

Genetic diversity of glucose phosphate isomerase from Entamoeba histolytica

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, II-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.     

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.