Biomedical subjects

Vivek Kapur

Publications and source records attributed to Vivek Kapur.

At least 37 records · Page 2Linked to original sources

PubMed2005

Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis.

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.

Bacterial Proteins↗

PubMed2005

Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures.

A liquid culture followed by molecular confirmation was evaluated for potential to improve sensitivity and reduce time to diagnosis of Mycobacterium avium subsp. paratuberculosis infection. Fecal samples from 240 animals from Ohio farms were assessed for presence of M. avium subsp. paratuberculosis using four different protocols: (i) sedimentation processing followed by inoculation on Herrold's Egg Yolk media (HEYM) slants (monitored biweekly up to 16 weeks), (ii) double centrifugation processing followed by inoculation on HEYM slants (monitored biweekly up to 16 weeks), (iii) liquid-solid double culture method using modified 7H9 broth (8 weeks) followed by subculture on HEYM slants (monitored up to 8 weeks), and (iv) liquid culture using modified 7H9 broth (8 weeks) followed by molecular assays for the presence of two M. avium subsp. paratuberculosis-specific targets. The number of positive samples detected by each protocol was 37, 53, 65, and 76, respectively. Twenty-seven samples were positive by all four methods. Based on samples positive by at least one method (n = 81), the sensitivities for sedimentation processing, double centrifugation processing, liquid-solid double culture, and liquid culture followed by molecular confirmation were 46%, 65%, 80%, and 94%, respectively. Fingerprinting of the positive samples using two polymorphic (G and GGT) short sequence repeat regions identified varying levels of within-farm and between-farm diversity. Our data indicate that liquid culture followed by molecular confirmation can significantly improve sensitivity and reduce time-to-diagnosis (from 16 to 8 weeks) of M. avium subsp. paratuberculosis infection and can also be efficiently employed for the systematic differentiation of M. avium subsp. paratuberculosis strains to understand the epidemiology of Johne's disease.

Bacterial Typing Techniques↗

PubMed2005

Amplified fragment length polymorphism to detect clonal diversity and distribution of Mycobacterium avium subspecies paratuberculosis in selected Minnesota dairy cattle.

The molecular ecology of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, is not well understood in the United States. In this study, a DNA fingerprinting method, amplified fragment length polymorphism (AFLP), was used to subtype the pathogen and assess the clonal diversity of MAP in Minnesota dairy herds. Fifty-six fecal culture test-positive isolates from various Minnesota counties and culture dates were analyzed in this study. The AFLP identified 11 profiles with 50% of isolates representing 1 major profile. The major profile was distributed across the state. The genetic diversity of bovine MAP clones in Minnesota based on AFLP analysis of this data appears to be relatively low.

Animals↗

PubMed2004

The genome of Cryptosporidium hominis.

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

Animals↗

PubMed2004

Identification and analysis of safener-inducible expressed sequence tags in Populus using a cDNA microarray.

Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra x Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 singletons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription-polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

Acetophenones↗

PubMed2004

Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells.

High throughput detection of differential expression of genes is an efficient means of identifying genes and pathways that may play a role in biological systems under certain experimental conditions. There exist a variety of approaches that could be used to identify groups of genes that change in expression in response to a particular stimulus or environment. We here describe the application of suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis for isolation and identification of chicken transcripts that change in expression on infection of host cells with a paramyxovirus. SSH was used for initial isolation of differentially expressed transcripts, a large-scale validation of which was accomplished by microarray analysis. The data reveals a large group of regulated genes constituting many biochemical pathways that could serve as targets for future investigations to explore their role in paramyxovirus pathogenesis. The detailed methods described herein could be useful and adaptable to any biological system for studying changes in gene expression.

Journal Article↗

PubMed2004

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

Animals↗

PubMed2004

Genomic-scale analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl cholera.

We have recently reported the gene expression profile of Pasteurella multocida during growth in the blood of chickens with fowl cholera. Here we report the gene expression profile of P. multocida during growth in the livers of similarly infected chickens. We compared expression profiles of bacteria harvested from the livers of infected chickens with late-stage fowl cholera with those of bacteria grown in rich medium. Independent analysis of bacterial expression profiles from three individual chickens indicated that 93 P. multocida genes were always differentially expressed during growth in liver tissue. Of these 93 genes, 49 were upregulated and 44 downregulated in the host. Many of the upregulated genes were involved in energy production and conversion (9/49) and carbohydrate transport and metabolism (8/49), and a number of these have been shown to be induced under anaerobic conditions in other species. The downregulated genes were generally of unknown or poorly characterised functions (14/44). Comparison of the differentially regulated gene sets identified for growth in liver with those identified previously for growth in blood allowed the identification of a core set of 13 upregulated and 16 downregulated genes that were differentially regulated in at least five of the six infections studied.

Anaerobiosis↗

PubMed2004

Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen.

Johne's disease, a chronic inflammatory disease caused by infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is one of the most prevalent and costly diseases of dairy cattle worldwide. This ruminant pathogen is closely related to the ubiquitous animal and human pathogen Mycobacterium avium subspecies avium (M. avium), confounding the development of specific diagnostic reagents. Exacerbating this problem further is that most existing microbiological, serological, and immunologic assays for the identification of infected animals are inadequate. This is primarily because of the slow-growing nature of the organism, genetic intractability and the previous lack of information on M. paratuberculosis subspecies-specific genes or proteins that may enable the development of specific and sensitive assays. New detection tools are critically needed to definitively answer questions surrounding M. paratuberculosis as a foodborne pathogen as well as aid in determining if it is a contributing factor in Crohn's disease. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratories has been a major step forward in meeting this need. We have performed studies that utilize genomic information for the identification of specific DNA sequences and protein antigens in M. paratuberculosis. Based on a preliminary in silico comparison of the M. paratuberculosis genome sequence with that of M. avium, we have now identified at least 35 novel coding sequences that are unique to M. paratuberculosis. These in silico data were then confirmed and expanded by PCR amplification analysis with DNA from several species and isolates of mycobacteria. Finally, these unique sequences have been incorporated into an antigen discovery project that may allow reliable detection of the bacterium in antigen-based diagnostic tests. Application of these new tools in addressing foodborne related issues of M. paratuberculosis is discussed.

Animals↗

PubMed2004

A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host.

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island.

ATP-Binding Cassette Transporters↗

PubMed2004

Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis.

The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium avium subsp. paratuberculosis. However, because of genetic similarity to other mycobacteria comprising the M. avium complex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. avium subsp. paratuberculosis that are not represented in any other mycobacterial species tested (n = 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol. 40:1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of these M. avium subsp. paratuberculosis-specific sequences in Escherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coli expression vectors encoding polyhistidine or maltose binding protein (MBP) affinity purification tags. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. avium subsp. paratuberculosis. These studies showed that 5 of the 21 gene products are produced by M. avium subsp. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same five M. avium subsp. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. avium subsp. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.

Animals↗

PubMed2004

Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.

We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.

Animals↗

PubMed2004

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species.

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.

Animals↗

PubMed2004

Characterization of novel coding sequences specific to Mycobacterium avium subsp. paratuberculosis: implications for diagnosis of Johne's Disease.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's Disease, an economically important intestinal ailment of ruminants. Due to the considerable genetic and serologic cross-reactivity with closely related and ubiquitous members of the M. avium complex, a species-specific method for the serological diagnosis of Johne's disease is unavailable. Computational and PCR-based analysis of the complete genome sequence of M. avium subsp. paratuberculosis led to the identification of 13 open reading frames with no identifiable homologs. One of these sequences is a putative insertion element present in six copies on the M. avium subsp. paratuberculosis genome. These novel M. avium subsp. paratuberculosis genes were cloned into Escherichia coli expression vectors, and nine were successfully expressed as recombinant fusion proteins. Five of these proteins were purified in sufficient amounts to allow immunoblot analyses of their reactivity with sera from naturally infected cattle as well as mice and rabbits exposed to M. avium subsp. paratuberculosis. Fusion proteins representing MAP0862, MAP3732c, and MAP2963c were recognized by nearly all of the sera tested, including those from cattle in the clinical stages of disease. Notably, further analysis of the protein encoded by MAP0862 showed that it reacted with sera from additional infected cattle but not with sera from uninfected control animals. The fusion product of MAP0860c did not react with any of the sera tested, while the remaining four proteins were variably recognized by sera from M. avium subsp. paratuberculosis-infected cattle. Collectively, the results of this study demonstrate the utility of genomic data to identify potential diagnostic sequences.

Animals↗

PubMed2004

Three novel herpesviruses associated with stomatitis in Sudan plated lizards (Gerrhosaurus major) and a black-lined plated lizard (Gerrhosaurus nigrolineatus).

Glossal stomatitis was observed in a Sudan plated lizard (Gerrhosaurus major) with severe dyspnea. On necropsy, intranuclear inclusion bodies were seen in the periglottal lingual epithelium. Labial stomatitis was seen in a second Sudan plated lizard and a black-lined plated lizard (G. nigrolineatus). Degenerate polymerase chain reaction (PCR) primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase gene were used to amplify products from lesions from each lizard. Nucleotide sequencing of the PCR products showed that the sequence from each lizard was unique. Phylogenetic and comparative sequence analyses suggest that these viruses are novel members of the subfamily Alphaherpesvirinae, and they are here termed gerrhosaurid herpesviruses 1-3. Results of our analyses suggest that the genus Gerrhosaurus can be infected by these novel herpesviruses.

Amino Acid Sequence↗

PubMed2003

MGView: an alignment and visualization tool to enhance gap closure of microbial genomes.

Gap closure is a challenging phase in microbial random shotgun genome sequencing projects, particularly since genome assemblies are often complicated by the presence of repeat elements, insertion sequences and other similar factors that contribute to sequence misassemblies. While it is well recognized that the conservation of genetic information between microbial genomes, combined with the exponential increase in available microbial sequences, can be exploited to increase the efficiency of gap closure, we lack the computational tools to aid in this process. We describe here a new tool, MGView, which was developed to create a graphical depiction of the alignment of a set of microbial contigs against a completed microbial genome. The results of our assembly of the Staphylococcus aureus RF122 genome show that MGView enables a considerable reduction in time and economic cost associated with closure. Together, the results also show that the application of MGView not only enables a reduction in fold-coverage requirements of the random shotgun sequence phase, but also provides interesting insights into differences in gene content and organization between finished and unfinished microbial genomes.

Base Sequence↗

PubMed2003

Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates.

BACKGROUND: Mycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study. RESULTS: To characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical. CONCLUSIONS: We present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Bacterial Proteins↗

PubMed2003

Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.

Systemic lupus erythematosus (SLE) is a complex, inflammatory autoimmune disease that affects multiple organ systems. We used global gene expression profiling of peripheral blood mononuclear cells to identify distinct patterns of gene expression that distinguish most SLE patients from healthy controls. Strikingly, about half of the patients studied showed dysregulated expression of genes in the IFN pathway. Furthermore, this IFN gene expression "signature" served as a marker for more severe disease involving the kidneys, hematopoetic cells, and/or the central nervous system. These results provide insights into the genetic pathways underlying SLE, and identify a subgroup of patients who may benefit from therapies targeting the IFN pathway.

Down-Regulation↗