Immunoaffinity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals.

The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies. The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine.

Triterpenoid saponins containing an acetylated branched D-fucosyl residue from Quillaja saponaria Molina.

Seven novel saponins were isolated from a bark extract of Quillaja saponaria Molina. the compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as quillaic acid substituted at C-3 with oligosaccharides consisting of various compositions of D-glucuronic acid D-galactose, D-xylose, and L-rhamnose and at C-28 with complex oligosaccharide structures consisting of various compositions of D-xylose, L-rhamnose, D-apiose and a branched 4-O-acetyl-D-fucose residue.

Triterpenoid saponins from Quillaja saponaria.

Three new saponins were isolated from a commercial bark extract of Quillaja saponaria Molina. These compounds were also obtained as degradation products from larger saponins in this extract when treated with strong alkali. The compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as quillaic acid 3-O-¿beta-D-galactopyranosyl-(1-->2)-beta-D-glucopyranosiduronic acid¿, 3-O-¿alpha-L-rhamnopyranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)] -beta-D-glucopyranosiduronic acid¿ and 3-O-¿beta-D-xylopyranosyl-(1-->3)-[beta-D-galactopyranosyl -(1-->2)]-beta-D-glucopyranosiduronic acid¿, respectively.

Protective effect of two Mycoplasma gallisepticum protein fractions affinity purified with monoclonal antibodies.

Two protein fractions of Mycoplasma gallisepticum (Mg) were affinity purified with monoclonal antibodies A3 and B3, and tested for protective capacity in chickens. One fraction, designated MgP1, appeared as a doublet of 64 and 62kDa bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, while MgP2 consisted of five polypeptides (64, 56, 47, 45 and 43 kDa). The molecular mass, haemagglutination activity and matching amino acid sequence of MgP1 suggest that it is identical to pMGA1.2, the putative haemagglutinin of Mg. Groups of Mg-free chickens were immunized once or twice with 1 or 5 mug MgP1 or MgP2, or a combination of the two, and adjuvanted with immunostimulating complexes. Except for the group given 1 mug MgP1, all vaccinated groups showed a significant (P < 0.01) reduction in air sac lesions after challenge compared with unvaccinated controls. MgP2 appeared more protective than MgP1. Vaccination twice with MgP2 was the only regime that produced a detectable serum antibody response.

A non-haemagglutinating isolate of mink enteritis virus.

A virus was isolated from mink showing clinical and pathological signs of mink enteritis. This virus was identified as mink enteritis virus (MEV) from results of serological tests, determination of its density in CsCl (1.415 g cm-3), and morphology, including size (20 nm in diameter). The isolate was designated MEV-S. In contrast to other known MEV strains, the MEV-S isolate has no haemagglutinating (HA) activity with swine red blood cells (RBCs) at 4 degrees C and pH 6.8. Neither was there any HA at other pH values and temperatures, or when horse, bovine and rhesus monkey RBC's were used.

Separation and structural analysis of some saponins from Quillaja saponaria Molina.

A fraction of saponins from Quillaja saponaria Molina, QH-B, was fractionated by consecutive separations on three different reverse-phase HPLC systems. Eight compounds were isolated and the structures of these were elucidated mainly by sugar analysis and NMR spectroscopy. The structures consisted of a quillaic acid substituted with two different trisaccharides at C-3, beta-D-Galp-(1-->2)-[alpha-L-Rhap-(1-->3)]-beta-D-GlcpA and beta-D-Galp-(1-->2)-[beta-D-Xylp-(1-->3)]-beta-D-GlcpA, and a tetra- or pentasaccharide at C-28, beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3)]-alpha-L-Rhap-(1--> 2)-beta-D-Fucp and beta-D-Apif-(1-->3)-beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3) ]-alpha-L- Rhap-(1-->2)-beta-D-Fucp. These compounds were further substituted with an acyl group either at O-3 or O-4 of the fucose residue, which is the sugar linked to C-28 of the quillaic acid.

Characterization and purification of Parafilaria bovicola antigens by chromatofocusing to enhance specificity in serodiagnosis.

A study was conducted to determine if the purification of Parafilaria bovicola antigens can increase the specificity of serodiagnosis of parafilariasis in enzyme-linked immunosorbent assay. Antigens released from adult worms of P. bovicola were separated by chromatofocusing on a polybuffer exchanger of the pH range 7.3-4.0 Polypeptide analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed the presence of four major polypeptides with MWs of 41, 36, 24 and 20 kDa. Additional biochemical characterization identified the 24- and 20-kDa polypeptides as hydrophobic glycoproteins. The chromatofocusing purification procedures were also applied for separation of a whole-worm extract. Again, the 41- and 36-kDa antigens were identified in separate peak fractions. Using ELISA, it was shown that the 41- and 35-kDa antigens were recognized by bovine antibodies specific for P. bovicola, but not by other sera collected from cattle infected by Onchocerca gutturosa, Onchocerca lienalis, Ostertagia ostertagi and Dictyocaulus viviparus. The serological evaluation strongly suggests that the 41- and 36-kDa antigens are P. bovicola specific.

Preparation and evaluation of the specificity of Parafilaria bovicola antigen for detection of specific antibodies by ELISA.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occurred. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.

Observations on the use of ELISA for detection of Babesia bigemina specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated to study the cause of the high level of background reactions which hinders the application of ELISA as a field diagnostic test for Babesia bigemina. Different blockers to improve the specificity of the ELISA were compared. THe use of soya milk (25%), gelatin (2.5%) and chicken serum (2%) did not significantly improve the specificity of the test. It was noted that the presence of fibrinogen contributed to the positive ELISA results more than the presence of B. bigemina specific antigen. This conclusion was confirmed by testing bovine fibrinogen as a host protein antigen in ELISA which strongly responded against B. bigemina positive control sera. It is suggested that application of ELISA for B. bigemina is still unreliable until a more purified Babesia-specific antigen or specific monoclonal antibodies are available.

Evaluation of a monoclonal blocking enzyme-linked immunosorbent assay for the detection of Mycoplasma gallisepticum-specific antibodies.

A monoclonal blocking enzyme-linked immunosorbent assay (blocking-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in poultry sera with the help of a peroxidase-labeled monoclonal antibody (MAb) recognizing an epitope of a 56-kilodalton polypeptide (p56) of MG. Immunoglobulins from undiluted MG-positive sera prevent the MAb conjugate from attaching to its specific binding site on p56, which results in no color development. The opposite result--a strong color reaction--was obtained after incubation with MG-negative sera (or when no serum was added before the MAb conjugate). Results were expressed in percent inhibition or ELISA titers. The blocking-ELISA detected 84.7% positive chickens in an experimentally infected flock and 72.6% of chickens in naturally infected flocks, whereas the hemagglutination-inhibition (HI) test was positive only with 68.4% and 48.6% of these serum samples, respectively. All HI-positive serum samples reacted positively in blocking-ELISA. Of sera negative by the HI test, 51.6% and 46.8% proved to be positive when examined with the blocking-ELISA. Overall agreement between the ELISA and HI test was 76.8%. Infection with closely related M. synoviae did not induce any false-positive reactions in blocking-ELISA. There was a strong positive correlation between HI and blocking-ELISA titers (r = 0.83).

Monoclonal antibodies to Mycoplasma gallisepticum membrane proteins.

Monoclonal antibodies (MAbs) were prepared to study the immunogenesis of Mycoplasma gallisepticum. Balb/c mice were immunized with M. gallisepticum immunostimulating complexes and the supernatant of heterokaryotes screened with M. gallisepticum and closely related M. synoviae as antigens in indirect enzyme-linked immunosorbent assay. All selected MAbs proved to be M. gallisepticum species-specific when they were tested against 10 different avian Mycoplasma species. After immunoblotting analysis, five polypeptides were identified with estimated molecular weights of 110,000, 66,000, 64,000, 56,000, and 50,000. Cell membrane localization of the recognized polypeptides was studied by immunoelectron microscopy. None of the MAbs inhibited the hemagglutinating activity of freshly prepared M. gallisepticum. However, one MAb (B3) specific for p56 agglutinated the stained M. gallisepticum antigen in the slide agglutination test. Results seemed to correlate with published information on the protein composition and agglutinating activity of Mycoplasma gallisepticum.

Four-year study of caries inhibition of intensive Duraphat application in 11-15-year-old children.

The purpose of the study was to evaluate the caries preventive effect and cost of an intensive application of Duraphat varnish, added to the regular preventive program for 11-15-yr-old children in a Swedish Dental Community Clinic. In 1987, the 134 11-yr-old children in Floda were divided into two groups, every second child to each. Children with fixed orthodontic appliances were excluded. The test group received three applications of Duraphat varnish during 1 week, once a year, by a dental nurse. The control group received one application at the annual check-up. Both groups were included in the regular preventive program at the clinic. The total time cost for the clinic was estimated and used to calculate the cost per hour for dentists and nurses. The caries increment and progression were estimated both by routine diagnosis and by a careful study of radiographs taken at the beginning and end of the study period. There was a small caries increment and progression in the test group as compared to the control group. The difference was statistically significant for all aspects studied. The costs were about the same in both groups but more time was used in the test group. The administrative effort for the staff was considerable for the intensive Duraphat application.

Equine herpes virus 1 (EHV-1) in liver, spleen, and lung as demonstrated by immunohistology and electron microscopy.

Ten aborted foals, diagnosed as infected with Equine Herpes Virus 1 (EHV-1) on histopathological criteria, were examined for the presence of EHV-1 using immunohistology as the investigative instrument. The primary reagent was an antiserum specific for viral envelope glycoproteins. Immunohistology localised EHV-1 to areas of liver necrosis and to the cytoplasm of infected Kupffer cells and hepatocytes. Cytoplasmic immunolabelling was also prominent in reticular cells of the red pulp of the spleen and in intact and degenerated bronchiolar epithelium. Cytoplasmic immunolabelling was seen in morphologically unchanged cells and in cells containing intranuclear inclusion bodies. Three aborted foetuses with no histological signs of EHV-1 infection were negative when immunostained for EHV-1. Detection by electron microscopy of EHV-1 virions confirmed the EHV-1 specificity of the immunolabelling procedure.

[Plasma-desorption mass spectrometry. Study of the desorption mechanism and prospects for use]. / Plazmenno-desorbtsionnaia mass-spektrometriia. Izuchenie mekhanizma desorbtsii i vozmozhnosti primeneniia.

Particle impact mass spectrometry and in particular the use of MeV particles as in plasma desorption mass spectrometry (PDMS) applied to biomolecules is described. Experimental and theoretical studies of the mechanisms involved for large molecular ion ejection are treated in some detail. Applications of PDMS mass spectrometry to proteins are discussed.

Dental caries and restorations in teenagers. I. Index and score system for radiographic studies of proximal surfaces.

An index and a score system for radiographical longitudinal studies of caries progression on proximal tooth surfaces are presented and compared with the DF-surface system. Generally, a good correlation between DF-surface differences and scores was found in a three-year study on teenagers. However, the score system seems to forward better means for a detailed study of the individual.

Dental caries and restorations in teenargers. II. A longitudinal radiographic study of the caries increment of proximal surfaces among urban teenagers in Sweden.

Within a group of 16 year old school children leaving the regular school dental care, the number of carious lesions and fillings in proximal tooth surfaces was radiographically studied, using an index system. Three years later, the same individuals were re-examined. The results showed that intact surfaces and surfaces with small carious lesions were more resistant to change than others. A minority of the individuals was found to be responsible for a majority of the lesions established during the three year interval. Generally, the progress of caries was slow.