[show abstract][hide abstract] ABSTRACT: The objective was to detect presence of calves excreting Mycobacterium avium subsp. paratuberculosis (MAP) in their feces as a consequence of being born to MAP fecal culture positive (vs. negative) dams. For each cow that was about to calf, approximately 10g of feces was collected manually by the herdsmen from the rectum using a disposable plastic examination sleeve within 48-72h prior to actual calving. Between 1 and 3d of birth, herd personnel collected approximately 10g of fecal samples followed by monthly visits to the farm at which time 10g of fecal samples were again collected by study investigators from each calf at approximately 30, 60 and 90d of age. Mycobacterium avium subsp. paratuberculosis was recovered from 8% (5/60) of the cows that gave birth to calves. However, MAP was not recovered from any of the fecal samples (0/240) collected from study calves. Findings of the present study suggest lack of evidence for fecal excretion of MAP in calves born to fecal culture positive (vs. negative) dams in a heavily infected herd.
Preventive Veterinary Medicine 12/2009; 93(2-3):242-5. · 2.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc(2)155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.
[show abstract][hide abstract] ABSTRACT: We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1-15) and 9 NA (1-9) subtypes of influenza A viruses of avian (n=16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.
Journal of virological methods 06/2009; 160(1-2):163-6. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle.
We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P < or = 0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals.
The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.
PLoS ONE 01/2009; 4(5):e5478. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains.
We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of approximately 230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs.
We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.
PLoS ONE 01/2009; 4(9):e7105. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10-40CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 10(1)-10(2)CFU S. Typhimurium/9mL rinsate, while in a recirculation format, detection limits were 10(2)-10(3)CFU/25mL rinsate. Reproducible detection at <10(1)S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.
Molecular and Cellular Probes 12/2008; 23(1):20-8. · 1.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. paratuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequence annotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have any annotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, 10-12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation, and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modus operandi for survival in the host.
Microbes and Infection 08/2008; 10(12-13):1274-82. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium avium (M. avium) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear.
A comparative genomic approach was used to identify large sequence polymorphisms among M. avium subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate M. avium subsp. paratuberculosis (MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. M. avium subsp. silvaticum isolates were observed to have a hybridization profile very similar to yet distinguishable from M. avium subsp. avium. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10.
Genome diversity in M. avium subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of M. avium were distinguishable by the presence or absence of specific polymorphisms.
[show abstract][hide abstract] ABSTRACT: In this study we investigated the ability of different Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains to survive in bovine monocyte-derived macrophages (MDMs) of cows naturally infected with M. paratuberculosis and control cows. We tested the hypotheses that infection status of cows affects macrophage killing ability and that survival of M. paratuberculosis in macrophages is dependent on the strain. Peripheral blood mononuclear cells (PBMC) were obtained from Johne's disease-positive (n=3) and age and stage of lactation matched Johne's disease-negative (n=3) multiparious cows. Following differentiation, MDMs were challenged in vitro with four M. paratuberculosis strains of different host specificity (cattle and sheep). Two hours and 2, 4, and 7 days after infection, ingestion, and intracellular survival of M. paratuberculosis strains were determined by fluorescence microscopy. There was no effect of the origin of MDMs (Johne's disease-positive or control animals) on phagocytosis, survival of bacteria, or macrophage survival. In contrast, important strain differences were observed. These findings suggest that some M. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens which may make it important to include strain typing when designing control programs.
Veterinary Immunology and Immunopathology 01/2008; 120(3-4):93-105. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms.
Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results.
Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry.
Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect.
Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.
American Journal of Veterinary Research 10/2007; 68(9):975-80. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.
Clinical and Vaccine Immunology 06/2007; 14(5):518-26. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johne's disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.
Clinical and Vaccine Immunology 02/2007; 14(1):102-5. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M. avium subsp. avium isolate, and they significantly up-regulated proinflammatory genes related to IL-6, T-cell receptor, B-cell receptor, and death receptor signaling within THP-1 cells. Additionally, we demonstrated consistency among infecting genotypes of M. avium subsp. paratuberculosis isolated from diverse hosts [cattle (n=2), human (n=3), sheep (n=2), and bison (n=1)] in quantitative reverse transcription-PCR analysis of seven differentially expressed genes. While the levels of expression induced by the bison isolate were different compared with cattle or human isolates, they followed the common anti-inflammatory, antiapoptotic trend. Our data suggest that the macrophage responses to M. avium subsp. paratuberculosis isolates from cattle and human sources, regardless of genotype, follow a common theme of anti-inflammatory responses, an attribute likely associated with successful infection and persistence. However, these expression patterns differ significantly from those in THP-1 cells infected with sheep isolates of M. avium subsp. paratuberculosis or the M. avium subsp. avium isolate. These data provide a transcriptional basis for a variety of pathophysiological changes observed during early stages of infection by different strains of M. avium subsp. paratuberculosis, a first step in understanding trait-allele association in this economically important disease.
Infection and Immunity 12/2006; 74(11):6046-56. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: To compare shedding patterns and serologic responses to bovine coronavirus (BCV) in feedlot calves shipped from a single ranch in New Mexico (NM calves) versus calves assembled from local sale barns in Arkansas (AR calves) and to evaluate the role of BCV on disease and performance.
103 feedlot calves from New Mexico and 100 from Arkansas.
Calves were studied from before shipping to 35 days after arrival at the feedlot. Nasal swab specimens, fecal samples, and serum samples were obtained before shipping, at arrival, and periodically thereafter. Bovine coronavirus antigen and antibodies were detected by use of an ELISA.
NM calves had a high geometric mean titer for BCV antibody at arrival (GMT, 1,928); only 2% shed BCV in nasal secretions and 1% in feces. In contrast, AR calves had low antibody titers against BCV at arrival (GMT, 102) and 64% shed BCV in nasal secretions and 65% in feces. Detection of BCV in nasal secretions preceded detection in feces before shipping AR calves, but at arrival, 73% of AR calves were shedding BCV in nasal secretions and feces. Bovine coronavirus infection was significantly associated with respiratory tract disease and decreased growth performance in AR calves.
Replication and shedding of BCV may start in the upper respiratory tract and spread to the gastrointestinal tract. Vaccination of calves against BCV before shipping to feedlots may provide protection against BCV infection and its effects with other pathogens in the induction of respiratory tract disease.
American Journal of Veterinary Research 09/2006; 67(8):1412-20. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: We analyzed the multilocus short sequence repeats (SSRs) of 211 and 56 isolates of Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium, respectively. The M. avium subsp. paratuberculosis isolates could be differentiated into 61 genotypes. The M. avium subsp. avium isolates showed limited diversity. These SSRs are stable and suitable for studying the molecular epidemiology of M. avium subsp. paratuberculosis.
Journal of Clinical Microbiology 09/2006; 44(8):2970-3. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (or paratuberculosis). Paratuberculosis is a chronic gastroenteritis mainly affecting cattle, sheep and other ruminants. MAP is also of concern due to the heretofore unresolved issue of its possible role in Crohn's disease in humans. We present here a review of MAP (i) mobile genetic elements; (ii) repetitive elements; (iii) single nucleotide polymorphisms; and (iv) whole-genome comparisons to study the molecular epidemiology of MAP. A summary of the findings to date is presented, and the discriminatory power, advantage and disadvantages of each of the methods are compared and discussed.
Microbes and Infection 05/2006; 8(5):1406-18. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recently, genetically diverse porcine noroviruses (NoV) and sapoviruses (SaV) were identified from field pig fecal samples. Reverse transcription (RT)-PCR is the primary method used for detection of human NoVs and SaVs. However, RT-PCR inhibitors frequently cause false-negative results. In this study, a competitive internal control (IC) RNA, specific for use in the SaV RT-PCR assay, was developed to monitor inhibition of RT-PCR; primers for detection of genetically diverse porcine NoVs and SaVs were designed; and microwell hybridization assays to confirm the specific RT-PCR products were developed. The primer pairs and the RT-PCR-hybridization combinations were compared using representative porcine NoV and SaV strains, positive pig fecal samples and a panel of 30 field pig fecal samples. Extracted RNA from 3 of 30 samples failed to amplify the IC RNA. However, this inhibition was not present after a 10-fold dilution of the extracted RNA. The five different RT-PCR-hybridization combinations developed specifically detected all three genotypes of porcine NoVs, all GIII porcine SaVs, unclassified JJ681-like, QW19 and LL26-like porcine SaVs, respectively. These RT-PCR-hybridization assays are specific, less time consuming and economical and particularly applicable to testing large number of samples for porcine NoVs and SaVs.
Journal of Virological Methods 04/2006; 132(1-2):135-45. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The identification of Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition. Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of Cryptosporidium parasites in environmental samples.
[show abstract][hide abstract] ABSTRACT: DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.
Experimental Biology and Medicine 03/2006; 231(2):204-14. · 2.80 Impact Factor