Use of synthetic oligonucleotide DNA probes for identification and direct detection of Bacteroides forsythus in plaque samples.

Oligonucleotide DNA probes complementary to the hypervariable region of the 16S rRNA of Bacteroides forsythus were tested for their specificity and sensitivity against reference and clinical isolates of 73 different species of bacteria. The probes were used to detect the organism directly from nucleic acid extracts of subgingival plaque samples, and the results were compared with results of detection by culture methods. The data demonstrate that the probes are very specific (100%) and sensitive (100% when they are compared with those obtained by the culture method) and could reliably detect the organism in plaque samples. When a probe to a conserved region of the 16S rRNA (UP9A) was used to estimate the quantity of bacteria present in plaque samples, it gave results comparable to those of culture methods. The data suggest that total microbial load may be a parameter in periodontal disease.

A versatile solid support system for oligodeoxynucleotide probe-based hybridization assays.

A procedure for immobilization of well-defined quantities of oligodeoxyribonucleotides (ODNs) to a versatile nylon support is described. The solid support, a nylon-6/6 bead, is covalently coated with poly(ethyleneimine) to provide a reactive spacer-arm for attachment of ODNs. 5'-Aminohexyl-tailed ODNs are selectively activated using 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) and then covalently attached to the bead via the triazine moiety. The modified nylon support has a low level of binding of nonspecific nucleic acid and efficiently captures both RNA and DNA targets.

Haptenated nylon-coated polystyrene plates as a solid phase for ELISA.

An ELISA system, based on the novel use of a hapten-nylon conjugate as solid-phase coating antigen, has been applied in the screening of hybridoma cultures for anti-hapten monoclonal antibodies directed against the herbicide atrazine and its derivatives. Conjugation of a 2-aminocaproic acid derivative of atrazine with DCC to polyamide (Nylon 6) gave haptenated nylon which was soluble in aqueous cresol-ethanol mixtures and adsorbed efficiently on polystyrene microtitre plates. Reproducible ELISA results were obtained with culture supernatants of hybridomas derived from spleen cells of mice that had been immunized with atrazine-bovine serum albumin conjugates. Satisfactory results were also obtained with a water soluble peptide conjugated to nylon for use as a coating antigen in an ELISA. Plates coated with hapten-nylon as antigen have the added advantage that they can be stored at room temperature for at least 6 months without loss of activity. Nylon therefore appears to have general applicability as a carrier for both non-polar and polar haptens in the preparation and use of coating antigens.

The presence of Cowdria ruminantium antigen in various tissues of Amblyomma hebraeum imagines as detected by an enzyme-linked immunosorbent assay.

Investigation into the presence of C. ruminantium antigen, using an enzyme-linked immunosorbent assay (ELISA) in various tick tissues and haemolymph of adult Amblyomma hebraeum ticks revealed that the organism invades a number of body parts and can be demonstrated in A. hebraeum. In females, the gut, salivary glands, hypodermis and synganglion and in males, the salivary glands and gut showed the highest concentration.

Kinetic properties of toxic protease inhibitors isolated from tick eggs.

1. Egg-toxins from Rhipicephalus evertsi evertsi, Boophilus microplus, Boophilus decoloratus and Hyalomma truncatum were found to be inhibitors of trypsin and in two cases also of chymotrypsin. 2. Fast tight-binding and slow-binding inhibition were observed. 3. Immunological identity of the toxins were assessed with Ouchterlony immunodiffusion and ELISA. 4. The protease content of B. decoloratus and Amblyomma hebraeum tick eggs were determined by a linked enzyme assay. 5. The predictive value of the kinetic constants in inferring a possible physiological role was discussed.

Purification of Cowdria ruminantium by density gradient centrifugation.

The isolation of Cowdria ruminantium by differential and isopycnic density gradient centrifugation is reviewed with special reference to the suitability of Percoll as density gradient medium. Infected sheep brain, Amblyomma hebraeum nymphae and various mouse organs were used as starting material. By these methods, partially purified viable populations of the organism with distinctly different densities were obtained. The conclusions are based upon results of analyses of density fractions by inoculation into sheep or mice, protein determination, electron microscopy and enzyme-linked immunosorbent assay. Morphological differences were observed in the density fractions obtained from infected brain tissue and A. hebraeum.

Purification of Cowdria ruminantium by lectin cellular affinity chromatography.

This review covers the isolation of Cowdria ruminantium by lectin cellular affinity chromatography from different Amblyomma hebraeum sources. Cellular affinity chromatography has been reviewed with special attention being given to the application of this technique in the isolation of rickettsiae.

Purification of Cowdria ruminantium by immunoadsorbent affinity chromatography.

Immunoselective methods with special reference to immunoadsorbent affinity chromatography as a means for the isolation of Cowdria ruminantium are reviewed. Attention is given to the source of the organism, immunization, purification of antibodies, coupling of antibodies to insoluble matrixes and desorption procedures.

Biochemical studies on the salivary glands and haemolymph of Amblyomma hebraeum.

The functional significance of some components of salivary glands and of their secretion and of haemolymph of Amblyomma hebraeum and other tick species is reviewed with respect to host responses at the attachment site, the survival of specific pathogens in the vector, the transmission of pathogens and immunological responses of the host to tick infestation.

Theoretical aspects of the enzyme-linked immunosorbent assay technique and its use in the detection of Cowdria ruminantium antigen and antibody in reacting animals.

Numerous parameters affect the enzyme-linked immunosorbent assay activity and the assay conditions must therefore be carefully controlled to obtain reproducible results. These parameters include temperature, pH, ionic strength, buffer composition, cofactors, substrate depletion, product inhibition, increasing reversal of reaction as product concentration increases, adsorption of enzyme to solid supports, denaturation and non-enzymatic background rate. An ELISA was used to detect Cowdria ruminantium antibodies during the course of heartwater disease. IgM antibodies reached a maximum on the 4th day after infection and disappeared on the 7th day. IgG antibodies first appeared on the 8th day and continued to increase during the remainder of the observation period of 28 days. The presence of C. ruminantium in the blood fractions of diseased animals was demonstrated by an enzyme-linked immunosorbent assay. The earliest detection of C. ruminantium antigen was in plasma and serum on the 4th day after inoculation. Of all the blood fractions investigated, the red blood cell fraction showed the highest concentration and this reached a maximum on the 12th day after infection.

Biochemical studies on the eggs of Amblyomma hebraeum.

The isolation, characterization and kinetic properties of a toxic and non-toxic anti-protease from the eggs of A. hebraeum are compared with similar data obtained for eggs of other tick species. The biochemistry of various physiological processes in tick eggs is reviewed.

The influence of the sesquiterpene lactones from Geigeria on mast cell degranulation.

The sesquiterpene lactones isolated from Geigeria were found to be incapable of inducing rat peritoneal mast cell degranulation at levels of 0.3-1.6 mM. The sulphydryl reagent, N-ethylmaleimide, too was unable to trigger mast cell secretion. Instead, it was observed that these compounds inhibited the release of histamine induced by Compound 48/80. Pretreatment of the lactones and N-ethylmaleimide with the amino acid, L-cysteine, reduced their inhibition ability of histamine release to a considerable extent, but not completely. Geigerin(V), which lacks an alpha-methylene group and the chemically prepared cysteine-adduct of dihydrogriesenin(I), were also capable of inhibiting mast cell secretion by Compound 48/80, but to a lesser extent.

The development of an ELISA-assay for semi-quantitative detection of dihydrogriesenin, a sesquiterpene lactone from Geigeria.

Certain species of Geigeria contain sesquiterpene lactones which cause vomiting disease in sheep. Dihydrogriesenin (DHG), a sesquiterpene lactone from G. asperta, contains an alpha-methylene function which can spontaneously react with thiol groups on proteins to form a covalent adduct. A specific antiserum against a DHG-protein adduct can be used to determine the fate of DHG in poisoned animals. The preparation of such an antiserum is reported in this paper. DHG was reacted with cysteine and subsequently coupled to serum albumin using the carbodiimide reaction. When rabbits were immunized with one such conjugate (DHG-bovine serum albumin), it was found that the carrier determinants were immunodominant. A DHG-specific anti-serum of sufficient (ELISA) titre could, however, be obtained by alternating serum albumin carriers for DHG in booster immunizations. The ELISA antigen-antibody reaction could be inhibited by prior reaction of the antisera with cysteinyl-DHG in solution.

Detection of Cowdria ruminantium antigen and antibody during the course of heartwater disease in sheep by means of an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was used to detect Cowdria ruminantium antibodies during the course of heartwater disease. IgM antibodies reached a maximum on the 4th day after infection and disappeared on the 7th day. IgG antibodies first appeared on the 8th day and continued to increase during the remainder of the observation period of 28 days. The presence of C. ruminantium in the blood fractions of diseased animals was demonstrated by an enzyme-linked immunosorbent assay. The earliest day of C. ruminantium antigen detection was in plasma and serum on the 4th day after inoculation. Of all the blood fractions investigated, the red blood cells showed the highest concentration, and this reached a maximum on the 12th day after infection.

Isolation of a neurotoxin from the salivary glands of female Rhipicephalus evertsi evertsi.

A quantitative study of the changes in the protein pattern of the salivary glands of female Rhipicephalus evertsi evertsi during the entire repletion process was undertaken. These results, in conjunction with the previously determined toxic phase, indicated the presence of a toxic protein. The development of a sensitive in vitro assay using a Xenopus nerve-muscle preparation, made it possible to identify toxic phases during feeding and to assay fractions of salivary gland extracts during toxin isolation. Sufficient amounts of electrophoretically and chromatographically homogeneous toxin could be obtained through the use of chromatofocusing, enabling its characterization with respect to molecular weight (68 kDa; determined by gel permeation chromatography), pI (6.00), and amino acid composition. The toxin was inactivated by pronase digestion as well as by antiserum.

Investigations into the function and chemical compositions of the porose areas secretion of Rhipicephalus evertsi evertsi during oviposition.

Major differences were observed in hexane, ethanol and butanol extracts of eggs obtained from Rhipicephalus evertsi evertsi females in which the porose areas functioned normally (AP+ eggs) and from R. evertsi evertsi in which the porose areas were selectively destroyed by electrocautery (AP- eggs). Mass yields and UV spectra of the hexane extracts were similar for AP+ and AP- eggs. The UV spectra changed only slightly in the 294-320 nm range with respect to time and temperature. High performance liquid chromatography revealed 2 components which originate from the porose areas. Mass spectroscopy of these components indicated the presence of aliphatic and phenolic groups. The ethanol and butanol extracts showed quantitative but no qualitative differences with respect to AP+ and AP- eggs. Electrophoretic fractionation of the butanol extracts revealed the presence of proteins in the secretion of the porose areas. Apart from this information on the chemical composition of the secretion, no indication was obtained of their function during oviposition of R. evertsi evertsi.

Isolation of Cowdria ruminantium by means of Percoll density gradient centrifugation and detection by an enzyme-linked immunosorbent assay.

The isolation of Cowdria ruminantium by means of Percoll density gradient centrifugation permits the recovery of partially purified viable populations of the organism possessing distinctly different densities. These conclusions are based upon results of analyses of density fractions by intravenous inoculation into sheep, protein determination, electron microscopy and enzyme-linked immunosorbent assay. Morphological differences were observed in the density fractions obtained from infected brain tissue and Amblyomma hebraeum nymphae.

The detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum by an enzyme-linked immunosorbent assay.

A sensitive and reliable enzyme-linked immunosorbent assay for the detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum nymphae is described. For the screening of antibodies, C. ruminantium from A. hebraeum nymphae, partially purified by wheat-germ lectin affinity chromatography, was used as antigen. To screen nymph populations, sera from either Ball 3 strain-infected sheep or Kumm-strain infected mice were used. By using appropriate controls the assays were rendered specific with respect to C. ruminantium.

The effect of the sesquiterpene lactones from Geigeria on glycolytic enzymes.

The effect of sesquiterpene lactones isolated from Geigeria was tested on three glycolytic enzymes. Phosphofructokinase was inhibited irreversibly by all of the sesquiterpene lactones, with ivalin(III) giving the highest extent of inhibition. Values for the kinetic constants Ki (1.3 mM) and kp (2.2 min-1) were established. Hexokinase and glyceraldehyde-3-phosphate dehydrogenase were also strongly inhibited at 1 mM and 3 mM concentrations of sesquiterpene lactones, respectively. Pre-incubation of ivalin with dithiothreitol decreased its inhibiting effect on phosphofructokinase, hexokinase and glyceraldehyde-3-phosphate dehydrogenase activities. Phosphofructokinase and hexokinase were protected against inhibition by ivalin by their respective substrates, adenosine-5'-triphosphate and glucose.

Isolation of Cowdria ruminantium by cellular affinity chromatography and detection by an enzyme-linked immunosorbent assay.

The isolation of Cowdria ruminantium by means of wheat germ lectin affinity chromatography as described in this paper permits the recovery of partially purified viable organisms under mild conditions in short time. These conclusions are based upon results of analyses of column fractions by intravenous inoculation into sheep, protein determination, electronmicroscopy and enzyme-linked immunosorbent assay. The entire purification procedure could be completed in 4-5 hours using only either infected sheep tissue or nymphae as starting material.