Genetic heterogeneity of Borrelia afzelii in the natural focus of the Middle Urals

As shown by sequencing the spacer rrf (5S)--rrl (23S) in 72 isolates of B. afzelii (one of the causative agents of Ixodes tick borne Borrelia infections) and the chromosomal gene coding protein P66 in 22 isolates, that in the natural focus located in the Middle Urals two different genetic subgroups (VS461 and NT28) of this genospecies simultaneously circulate. These subgroups are represented by 5 gene variants (rrf) 5S--(rrl) 23S and 5 allelic variants in gene p66. The latter, similarly to spacer gene variants, are not linked with a definite host and occur in different rrf--rrl variants of the infective agent. At the same time the definite species of vectors and carriers may be the host of several different B. afzelii variants, both in the spacer and in the gene coding protein P66, which maintains the genetic heterogeneity of B. afzelii population in the natural focus.     

Difference in amino acid sequences of B. afzelii P66 surface-exposed loop region

In the present work, we performed a phenotyping analysis of 45 B. afzelii 89-a.a. long amino acid sequences of 7 different allele variants, corresponding to the surface-exposed loop region of P66. 45 investigated isolates showed 5 phenotypically different variants; 2 phenotypically different variants of loop region, in particular, also showed mutations in the putative monoclonal antibody H1337 binding site; the similarity between the amino acid sequences taken from different variants is about 96.66% to 98.88%; in one natural locus up to 3 different phenotypes of P66 could circulate simultaneously.     

Genetic variants of Borrelia afzelii, a pathogen of the ixodes tick borrelioses

Borrelia afzelii is one of two most important pathogens of the ixodes tick borrelioses (ITB) in Russia and neighboring countries. This pathogen circulates in various ecosystems and has a wide range of reservoir hosts and transmitters. The results of studies of genetic heterogeneity of the spirochaetae B. afzelii are considered. A total of 139 primary isolates were studied. The isolates were isolated from three species of Ixodes ticks at different stages of development and obtained from the laboratory of infection transmitters, Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Russran Academy of Medical Sciences, Moscow. The transmitters and reservoir hosts of borrelias were caught in natural foci of Russia (from Kaliningrad region irk the west to south Sakhalin in the east), Czechia, Lithuania, Estonia, Ukraine, and Moldavia. Analysis of genotype sequence similarity obtained by sequencing of the rrf(SS)-rrl(23S) spacer demonstrated that the B. afzelii genospecies incorporated no less than 10 genetic variants of spirochaetae, most variants being geographically widespread.     

Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia

Abstract A 66-kDa protein (p66) associated with the outer membrane of Lyme disease Borrelia was analysed at the molecular level. The chromosomal genes encoding p66 in B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90 were sequenced. Database searches revealed that the p66 gene sequences were homologous to a previously reported gene fragment of unknown function. The deduced amino acid sequences of p66 in different Lyme disease borreliae were 92–94% identical and had no homologs in the databases. Proteolytic cleavage patterns of p66 and a computer-predicted single trans-membrane helix suggested the presence of surface-exposed epitopes on the C-terminus.     

Comparative Studies on Borrelia afzelii Isolated from a Patient of Lyme Disease,Ixodes persulcatus Ticks,and Apodemus speciosus Rodents in Japan

The genospecies Borrelia afzelii was isolated from a patient of Lyme disease in Hokkaido, Japan, for the first time, by culturing the minced erythema lesion in BSK II medium. Two analytical methods, rRNA gene restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) using the specific primer set to amplify the 16S rRNA gene, revealed that this clinical isolate belongs to the group of B. afzelii. In our culture collection of spirochetes, part of the isolates from Ixodes persulcatus ticks, and from Apodemus speciosus rodents, were also classified as B. afzelii. These results strongly suggest that the agent pathogenic to humans is maintained in “rodent-tick” transmission cycle.     

Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange.

Analysis of the ospD gene has revealed that this gene is not universal among Lyme disease spirochete isolates. The gene was found to be carried by 90, 50, and 24% of the Borrelia garinii, B. afzelii, and B. burgdorferi isolates tested. Size variability in the ospD-encoding plasmid was also observed. Sequence analysis has demonstrated the presence of various numbers of a 17-bp repeated sequence in the upstream control (promoter) region of the gene. In addition, a region within the coding sequence where various insertions, deletions, and direct repeats occur was identified. ospD gene sequences from 31 different isolates were determined and utilized in pairwise sequence comparisons and construction of a gene tree. These analyses suggest that the ospD gene was the target of several recombinational events and that the gene was recently acquired by Lyme disease spirochetes and laterally transferred between species.     

Distribution of twelve linear extrachromosomal DNAs in natural isolates of Lyme disease spirochetes

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete, Borrelia burgdorferi (sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the B. burgdorferi type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the B. burgdorferi B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents Borrelia garinii and Borrelia afzelii; however, some of these plasmids had uniform but substantially different sizes from their B. burgdorferi counterparts.     

Allelic polymorphism of the tem(M) determinant in Mycoplasma hominis and Ureaplasma urealyticum clinical isolates resistant to tetracyclines

Of the 130 clinical isolates of Mycoplasma hominis from patients with nonspecific inflammatory diseases of the urogenital tract (UGT), approximately 10% contained the tet(M) gene after the course of treatment with tetracyclines. This gene was found in nine (25%) of the 36 Ureaplasma urealyticum clinical isolates. The nucleotide sequence of 13 tet(M) genes in TcR clinical isolates of M. hominis and five genes in U. urealyticum TcR clinical isolates was determined. A comparison of nucleotide sequences of eight tetM genes of different origin and tet(M) genes of Gardnerella vaginalis and M. hominis and U. urealyticum clinical isolates showed that the mosaic structure of the tet(M) gene is completely identical in 11 of 13 M. hominis TcR isolates but belongs to an unidentified allele different from those described earlier, Another new allelic variant of tet(M) was found in two isolates. In three of five TcR clinical isolates of U. urealyticum, a tet(M) gene, whose mosaic structure was identical to that of tet(M) reported previously for ureaplasmas, and also two new allelic variants, which have not been described so far, were found.     

Determination of genetic variability among the isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">anisopliae</Emphasis> from different geographical origins

The entomopathogenic fungus Metarhizium anisopliae is currently used as an efficient biological control agent against different insects. The prevalence and genetic variability of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae in Asian countries (China, Laos, Singapore and South Korea) and a European country (The Netherlands) was examined. The fungus was found to be widespread in agricultural and forest soils throughout China especially in the south and south western regions, with a maximum recovery percentage of 81.6, while in Laos it was found to be abundant in the forest soils only, the soil of Netherlands also seemed to be an excellent source of entomopathogens with a significant recovery of insect pathogenic fungi. Simple sequence repeats (SSR) and ITS-rDNA sequence data showed that the isolates are closely linked to each other. The gene diversity (He) and polymorphic information content values of sixty two isolates showed mean values of 0.37 and 0.63. The majority of the isolates belonged to one of the closely related genotypes and these were found to be dominant in the agricultural as well as forest ecosystem. Genetic distances among the isolates according to locations and different sources showed minimal variation ranging from 0.23 to 0.34%, with maximum genetic distance of 0.34% among the isolates from Laos and The Netherlands. The reason for the limited variation is uncertain, however even the similarity index among the isolates can endow with sufficient knowledge for the implications of the methods to develop this fungus as a microbial control agent.     

Genetics factors determining predisposition to chronic course of virus hepatitis and fibrosis in liver

Polymorphic variants of several genes IL4 C(-590)T, IL4RA Ile50Val, TNF G(-308)A were studied for their association with extent of the disease chronization which is marked by hepatic fibrosis stage. Gradual decrease in A allele frequency of polymorphic marker G(-308)A in TNF gene, from patients with weak fibrosis to patients with cirrhosis. Group of patients with weak fibrosis was characterized by higher frequency of A allele (24.5%) comparing with patients with moderate and pronounced fibrosis (13.4%) and cirrhosis (8.7%). Differences in heterozygous genotype frequencies of IL4 C(-590)T were found between patients with cirrhosis (68.2%) and groups of patients with moderate and marked fibrosis (39.1%).     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.     

Genetic characterization of pathogenic Borrelia, group A14S, isolated in Ukraine

Borrelia Ir-5215, isolated from ticks Ixodes ricinus in Ukraine (the Crimean autonomous region), was identified by the method of the polymorphism of the fragment length of the restriction amplicon of rRNA spacer region 5S-23S. Its Msel-restriction profile was relatively similar to that of B. afzelii. The sequencing of spacer region rrf (5S)-rrl (23S) and 16S rRNA gene, as well as the analysis of the similarity of nucleotide sequences, obtained in the course of these study, revealed the differences between Borrelia sp, lr-5215 and six European species of Borrelia burgdorferi sensu lato and a high level of similarity (more than 95.1% for 5S-23S rRNA and 99.4% for 16S rRNA gene) to three known representatives of genome group A14S (Borrelia spp. A14S, I-77 and PC-Rq17). This suggests that isolated Borrelia lr-5215 is a new representative of pathogenic B. burgdorferi sensu lato genome group A14S, which is spread, together with Central Europe, also in southern Ukraine.     

ARSB gene variants causing Mucopolysaccharidosis VI in Miniature Pinscher and Miniature Schnauzer dogs

Mucopolysaccharidosis (MPS) VI is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase, also called arylsulfatase B (ARSB, EC 3.1.6.12). Dogs with MPS VI show progressive predominantly oculoskeletal signs homologous to those in human and feline patients. We report herein two pathogenic ARSB gene variants in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI and a genotyping survey in these breeds. All exons and adjacent regions of the ARSB gene were sequenced from three affected Miniature Pinschers and three affected Miniature Schnauzers. Allelic discrimination assays were used for genotyping. A missense variant (NM_001048133.1:c.910G>A) was found in exon 5 of MPS VI-affected Miniature Pinschers that is predicted to result in a deleterious amino acid substitution of a highly conserved glycine to arginine (NP_001041598.1:p.Gly304Arg). In MPS VI-affected Miniature Schnauzers, a 56 bp deletion (NM_001048133.1:c.-24_32del) was found at the junction of exon 1 and its upstream region, predicting no enzyme synthesis. All clinically affected Miniature Pinschers and Miniature Schnauzers were homozygous for the respective variants, and screened healthy dogs in each breed were either heterozygous or homozygous for the wt allele. Whereas the Miniature Pinscher variant seemed to occur commonly (0.133 allele frequency), the Miniature Schnauzer variant was presumed to be rare. In conclusion, two breed-specific pathogenic ARSB gene variants were identified in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI, allowing for genotyping and informed breeding to prevent the production of affected offspring.     

Identification of adenovirus 7h heterogeneity in the E3 region.

Adenovirus genotype 7h was previously reported to be originated from a recombination event between adenovirus genotypes 7p and 3p. Based on those findings, further characterization of other adenovirus 7h strains become important to determine whether all adenovirus 7h strains arose from a single recombinational event. To explore such a possibility, 160 clinical isolates were studied after developing a PCR assay using a primer set designed to amplify the region corresponding to E3-7,7 Kd of adenovirus ADV 7p and E3-9 Kd of adenovirus 3p. The assay was able to differentiate most of the subgenus B strains from adeno 7h with the genotype 3d. The study of several adenovirus 7h clinical isolates revealed the existence of three variants of adeno 7h. One of the variants, 7h3, shows a high degree of similarity with gene E3-9 Kd of ADV 3p, but lacks the corresponding AUG codon. Our results suggest that more than one recombination event may explain the detection of three different types of adenovirus 7h. The genotype variants of adeno 7h were detected in different years, indicating that the recombination events took place independently from each other. The study of the recombination region may allow further understanding of the function of several viral polypeptides in the immune response, and understanding the mechanism involved in virulence associated to adenovirus 7h.     

Functional variants in the lipoprotein lipase gene and risk cardiovascular disease.

The current report is a quantitative review of the relationship between lipoprotein lipase gene variants and cardiovascular disease based on published population-based studies. Sixteen studies, representing 17,630 individuals, report allelic distribution for lipoprotein lipase gene variants among patients and control individuals. Patient outcomes included clinical cardiovascular disease events, documented coronary disease based on angiography, or intimal media thickening by B-mode ultrasonography. Mantel-Haenszel stratified analysis was used to compute a summary odds ratio and 95% confidence intervals for the association between rare allele in the lipoprotein lipase gene and disease status. Because of potential differing effects associated with different lipoprotein lipase variants, each lipoprotein lipase mutant allele was considered separately. The lipoprotein lipase D9N/-93G to T allele has a summary odds ratio of 2.03 (95% confidence interval 1.30-3.18), indicating a twofold increase in risk of coronary disease for carriers with this allelic variant. The summary odds ratio for the relationship of the rare lipoprotein lipase G188E variant with cardiovascular disease is 5.25 (95% confidence interval 1.54-24.29). The lipoprotein lipase N291S allele is associated with a marginal increase in cardiovascular disease (summary odds ratio 1.25, 95% confidence interval 0.99-1.60, P = 0.07). However, there is stronger evidence for a positive association in certain populations. The summary odds ratio for lipoprotein lipase S447X allele is 0.81 (95% confidence interval 0.65-1.0), which indicates a cardioprotective effect of this lipoprotein lipase gene variant. Thus, lipoprotein lipase gene variants are associated with differential susceptibility to cardiovascular disease.     

Sequence analysis of partial toxR gene from Philippine Vibrio isolates and design of toxR-targeted primers for detection

Vibriosis in penaeid species cultured in the Philippines results in massive mortalities and consequently in severe economic losses in the shrimp industry. Rapid and accurate detection of the causative agent of the disease is imperative. In this study, toxR gene sequence analysis of ten Vibrio isolates (from several provinces of the Philippines) implicated in disease affecting the penaeid shrimp (Penaeus monodon) was performed in order to develop a toxR-targeted PCR detection of similar strains of shrimp pathogens. Analysis of the partial toxR gene revealed 97-100% sequence similarity among the ten Philippine Vibrio isolates. Distinct sequence variation of the toxR gene, however, was observed between the Philippine Vibrio isolates and the type strains, with the Philippine isolates exhibiting only 92-93% and 74-75% sequence similarity with the type strain V. campbellii (NBRC 15631T) and V. harveyi (NBRC 15634T), respectively. The use of a PCR primer set that was designed based on toxR sequences of the Philippine Vibrio isolates amplified the expected 226-bp toxR fragment using templates from all ten Philippine Vibrio isolates. No amplified product was observed in PCR using templates from type strains of V. harveyi, V. campbellii, and other non-target bacteria, suggesting that the primers were specific for the Philippine Vibrio isolates. The toxR-targeted PCR primers reported in this study could be useful in the detection of Philippine Vibrio isolates associated with mortalities in the shrimp industry, which could not be detected in PCR using primers designed for type strains of V. harveyi and V. campbellii.     

Sexual mating of Botrytis cinerea illustrates PRP8 intein HEG activity

Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/?). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/? heterozygote, the homing endonuclease initiates intein ‘homing’ by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.