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PubMed2003

PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle.

A single polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used to characterise all important bovine trypanosome species. This is the first report of a sensitive pan-trypanosome PCR assay amplifying all species including T. vivax to a comparable extent using a single primer pair. A semi-nested PCR approach resulted in the detection of one T. congolense trypanosome genome/40 microl of blood, applied as buffy coat on filter paper. Restriction enzyme analysis using Msp1 and Eco571 gave a clear distinction between T. congolense, T. brucei, T. vivax and T. theileri. Several subgroups within the T. congolense group could be distinguished but no differences between the species belonging to the subgenus Trypanozoon or between T. simiae and T. theileri could be found. The use of MboII restriction enzyme allowed differentiation between T. simiae and T. theileri. The potential of the essay to be used as a suitable diagnostic tool is discussed.

Animals↗
PubMed1990

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis.

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.

Animals↗
PubMed1990

Lipophosphoglycan and secreted acid phosphatase of Leishmania tropica share species-specific epitopes.

Several species-specific monoclonal antibodies (T11, T13-T15) which only react with Leishmania tropica, recognize phosphorlated carbohydrate epitopes on lipophosphoglycan and the structurally related molecule, phosphoglycan, which is shed by promastigotes into spent culture medium. During immunoaffinity isolation of [32P]orthophosphate-labeled phosphoglycan on monoclonal antibody T15 conjugated to Sepharose 4B, a high-Mr component (approx. 200,000) was co-purified. The latter material is metabolically labeled with [35S]methionine and [3H]glucosamine. This glycoprotein was separated from phosphoglycan by chromatography on lentil lectin resin. The glycoprotein exhibited a L-tatrate-sensitive acid phosphatase activity, typical of secreted acid phosphatase (EC 3.1.3.2) from Leishmania. Monospecific antibodies to Leishmania donovani-secreted acid phosphatase selectively precipitated the L. tropica enzyme from immunoaffinity purified mixtures of the two antigens, and monoclonal antibodies to lipophosphoglycan precipitate the pure enzyme. Species-specific monoclonal antibodies to L. major lipophosphoglycan also recognized both L. tropica antigens. Treatment of the acid phosphatase with periodate or phosphodiesterase I abolished binding by the monoclonal antibodies to the pure enzyme. These results demonstrate that the two major secreted glycoconjugates of Leishmania tropica, the lipophosphoglycan and the acid phosphatase, share species-specific phosphorylated carbohydrate epitope(s).

Acid Phosphatase↗
PubMed1994

Pheromonal composition of two species of African Amblyomma ticks: similarities, differences and possible species specific components.

Two species of bont ticks, Amblyomma hebraeum and Amblyomma variegatum, common to Africa were studied to compare types and quantities of compounds known or believed to serve as components of the attraction-aggregation-attachment pheromone (AAAP). A complex of attraction, aggregation and attachment stimulating pheromone components are used by these ticks to detect hosts, mates and, perhaps, minimize interspecific breeding. Solvent extraction of pheromone emitting ticks followed by gas chromatography and mass spectrometry revealed little qualitative difference in the composition of the AAAP in these two species. However, subtle differences in the relative proportions of the pheromonal blend are noted suggesting that such differences in concentration may facilitate species-specific discrimination during aggregation and attachment. Differences in the relative abundance of benzaldehyde and methyl salicylate in the males of the two species were especially noteworthy. Possible means by which such differences in phenolic compound composition may affect the behavior of these ticks are discussed.

Animals↗
PubMed2004

Accurate identification of closely related Dendrobium species with multiple species-specific gDNA probes.

About 63 species of Dendrobium are identified in China, making the identification of the origin of a particular Dendrobium species on the consumer market very difficult. We report evaluation of multiple species-specific probes screened from genomic DNA for closely related Dendrobium species identification, based on DNA array hybridization. Fourteen species-specific probes were screened from five closely related Dendrobium species, D. aurantiacum Kerr, D. officinale Kimura et Migo, D. nobile Lindl., D. chrysotoxum Lindl. and D. fimbriatum Hook., based on the SSH-Array technology we developed. Various commercial Dendrobium samples and unrelated samples were definitely identified. The specificity and accuracy of the multiple species-specific probes for species identification was assessed by identifying various commercial Dendrobium samples (Herba Dendrobii). Hybridization patterns of these multiple probes on digested genomic DNAs of Dendrobium species indicated that there are distinct polymorphic sequence fragment in the higher eukaryotes. This is the first report on detection and utilization of multiple species-specific probes of Dendrobium in whole genomic DNA, and this could be useful tools not only for a new technical platform for the closely related species identification but also for epidemiological studies on higher eukaryotes.

DNA↗
PubMed2001

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy.

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.

Animals↗
PubMed2001

Subunit compositions of crustacean haemocyanins are species-specific: evidence from non-decapod species.

Electrophoretic examination of dissociated haemocyanin subunits from a number of amphipod, decapod and isopod crustaceans supports the hypothesis that subunit composition is species-specific, despite marked within-species variation in many species. General patterns of heterogeneity on native PAGE gels were also evident between groupings within the Amphipoda. Gammarid amphipods could be split into two groups; one characterised by a high degree of heterogeneity and the other by a low degree of heterogeneity. The talitrid amphipods generally displayed a low degree of heterogeneity similar to, although still distinct from, the second gammarid category. Haemocyanin from the Hyalidae, a family allied to the talitrids was highly heterogeneous, similar to the first gammarid group and unlike the talitrids. Isopod haemocyanin banding patterns were more similar to one another than to any of the amphipod or decapod species examined. In general, the molecular weights of the amphipod Hcs tended to be greater than those of the isopods, with the decapods being lowest of all. It is suggested that Hc subunit heterogeneity may be a useful tool for investigating speciation and speciation events, and for reliably separating very closely-related species (e.g. Gammarus spp.), purely on the basis of their Hc subunit compositions.

Animals↗
PubMed1994

Comparison of genus- and species-specific probes for PCR detection of mycobacterial infections.

We describe experiments comparing use of different DNA probes to detect mycobacteria in clinical specimens after PCR. The objective was to assess correlation between results using Mycobacterium genus-specific, and species-specific M. tuberculosis probes. Given sufficient concordance, sequential use of such probes would provide a useful screening tool. An evaluation of genus-specific probes compared use of repetitive sequences in the clone pMAv17 with the 65-kDa sequences. Sensitivity was 100% for pMAv17, 93% using the 65-kDa sequence; specificity was 70% for both. We then compared M. tuberculosis-specific probes developed by us (Tb400) with IS6110 and mpt40. Sensitivity using Tb400 was 100%; using IS6110 was 97%, and using mpt40 was 50%. Specificity using Tb400 and IS6110 was 68%, and was 70% using mpt40. Fourteen specimens which were PCR-positive and culture-negative, were positive using both genus probes, and the M. tuberculosis-specific probes Tb400, and IS6110. Ten of these were positive using mpt40.

Base Sequence↗
PubMed1998

Species-specific plasmid sequences for PCR identification of the three species of Borrelia burgdorferi sensu lato involved in Lyme disease.

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.

Base Sequence↗
PubMed1990

Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies.

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.

Animals↗
PubMed1987

Demonstration of species-specific sensitization to major allergens of Dermatophagoides species by solid-phase absorption of human IgE antibodies.

Twenty-five mite-allergic patients in the Brussels area were tested with the skin-prick test (1, 5 and 10 histamine equivalents by prick test (HEP] and RAST with standardized extracts of Dermatophagoides pteronyssinus (Dp) and D. microceras (Dm). Highly significant correlations (0.69 less than rS less than 0.96) between the two species coincided with marginal, but highly significant (P less than 0.01), greater reactions to Dp in all comparisons. Species-specific IgE antibodies to antigen Dp-42 (= P1) and Dp-X were readily detected by solid-phase serum absorption with heterologous antigen and subsequent crossed radioimmunoelectrophoresis (CRIE). An absorption single radial immunodiffusion (SRID) technique, involving major allergens Dp-42, Dm-6 and Df-6 of Dp, Dm and D. farinae (Df), demonstrated the presence of IgE antibodies reacting with epitopes common to all species, epitopes common to two species, absent on the third, and epitopes strictly specific for Dp. The majority of patients with a strong IgE binding reacted to both common and strictly species-specific epitopes. The serological findings were in good agreement with the exposure situation in the area: by species-specific ELISA for dust allergen content, Dp occurred as the dominating species, 20 out of 24 homes having dust levels of Dp-42 above 30 ng/g. Five and four homes had detectable levels of Dm-6 and Df-6, respectively, in dust.

Adolescent↗
PubMed2002

Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5' nuclease PCR assays.

This study describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi and E.intestinalis. The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10(4) and 10(-1) spores per PCR sample. The coefficients of variation were < or =5.2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10(4) spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.

Animals↗
PubMed1991

The genetic basis of a species-specific character in the Drosophila virilis species group.

The genetic basis of the species-specific dorsal abdominal stripe of Drosophila novamexicana was examined. The dorsal stripe is present in D. novamexicana and absent in all other members of the Drosophila virilis species group. Interspecific crosses between D. novamexicana and genetically marked D. virilis revealed that all four of the autosomes (except the tiny dot chromosome, which was not marked) and the sex chromosomes (the X and Y chromosome effects could not be disentangled) showed a significant effect on the width of the dorsal stripe. All the autosomes act approximately additively; only minor interactions were detected among them. No significant maternal effects were found. This means that a minimum of five loci are involved in the character difference between the two species, and this is the maximum number that this technique could discern. These results suggest that, based on the number of factors involved in the character difference, the inheritance of this character should be considered polygenic, but because chromosome 2 (the largest chromosome in the species) contributed over half of the variance toward the character difference, it is best to consider the inheritance oligogenic based on effect. The implications of these findings are discussed in light of the importance of macromutation in speciation and the sex chromosome theory of speciation.

Analysis of Variance↗
PubMed1991

The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.

At least two species-specific gene products are required for signal transduction by interferon gamma (IFN-gamma). The first is the IFN-gamma receptor, which binds ligand with high affinity in a species-specific manner. The second is an undetermined species-specific signal transducer(s). To determine whether the human IFN-gamma receptor (hIFN-gamma R) interacts directly with this signal transducer(s) and, if so, with what functional domain(s), we constructed expression vectors for the hIFN-gamma R and three hybrid human-murine IFN-gamma receptors. The hybrid receptors contained the extracellular, human IFN-gamma (hIFN-gamma) binding domain of the hIFN-gamma R, either the human or murine transmembrane domain, and either the human or murine intracellular domain. The vectors encoding these receptors were stably transfected into two mouse cell lines, one of which (SCC-16-5) contains a single copy of human chromosome 21. The resulting cell lines were treated with hIFN-gamma, and murine major histocompatibility complex class I antigen expression was analyzed by immunofluorescence flow cytometry. All transfected cell lines lacking human chromosome 21 remained insensitive to hIFN-gamma. However, all four of the IFN-gamma receptors were able to signal when expressed in the cell line containing human chromosome 21. We conclude that the extracellular domain of the IFN-gamma receptor is involved not only in the species specificity of IFN-gamma binding but also in signalling through interaction with an as yet unidentified species-specific factor(s) encoded by a gene(s) on human chromosome 21.

Animals↗
PubMed2002

Detection of Rhodococcus equi by polymerase chain reaction using species-specific nonproprietary primers.

Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10-base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.

Actinomycetales Infections↗
PubMed1992

Differentiation between thermophilic Campylobacter species by species-specific antibodies.

The four species of thermophilic campylobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari, are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.

Absorption↗
PubMed2002

Analyses of Streptococcus mutans in saliva with species-specific monoclonal antibodies.

Three species-specific monoclonal antibodies (MAbs) against Streptococcus mutans were used to detect and quantify S. mutans levels in saliva. This study shows that MAb-based salivary S. mutans tests exhibit significantly higher specificity and sensitivity than the commonly used selective culture method. Examination of nearly 2,000 human saliva samples shows that S. mutans counts in human saliva vary from less than 10,000 to a high 36 million cells/mL. Over 15% of the saliva samples examined have salivary S. mutans counts over 500,000 cells/mL. When saliva samples were collected at different time points during a day, the number of salivary S. mutans in the same human subject varied, especially before and after sugar uptake. Additionally, data obtained from stimulated versus unstimulated saliva in the same human subjects differed greatly and appear to be completely uncorrelated. This study provides useful information and tools for analyzing the role of S. mutans in human dental caries.

Antibodies, Bacterial↗