Agar Gel Diffusion Test Research Articles

Preliminary observations on the presence of visna-maedi in Italy.

The presence of visna-maedi in Italy is reported for the first time. History, clinical findings and gross and microscopic lesions typical of the disease were observed in two sheep flocks in Central Italy. Affected animals were afebrile, lacked energy, lost condition and had progressively worsening dyspnoea which ended fatally. A few showed locomotor disturbances but remained alert. Anaemia and leukopenia were present. The lungs were affected with fleshy consolidation. Histologically, the pulmonary lesions included bronchiolar epithelial hyperplasia, peribronchiolar lymphoid cuffing, alveolar epithelialization and very marked interstitial changes. In agar gel diffusion tests on sera from 106 animals from the two flocks, 44 were positive. Furthermore, when the same test was conducted as a preliminary screening procedure on 682 animals randomly chosen from 94 flocks in five Regions, 93 positive animals were revealed in 39 flocks, involving all of the Regions. The infection is likely to be widespread in Italy and to constitute a problem in at least some flocks or areas.

Comparison of agar-gel diffusion tests, counterimmunoelectrophoresis and enzyme-linked immunosorbent assay in the sera of skin test positives for paragonimiasis

Agar-gel diffusion test (AGD), counterimmunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay(ELISA) were examined with the sera of skin test positives for paragonimiasis. The crude antigen(Paragonimus whole worm extracts: protein concentration, 7.56mg/ml) and human sera were used in AGD and CIEP. And in ELISA test, diluted antigen with 1:40,000 of crude antigen and diluted sera with 1:100, 1:200 were used in the test. The positive identical ratio between AGD and CIEP reactions is 98 % and negative identical ratio is 100 %. One or three precipitin bands are observed in AGD. One to seven precipitin bands are also revealed in CIEP. Especially, deeply stained bands are observed in CIEP than those of AGD. The positive identical ratios between AGD and ELISA tests are 96 % in 1:100 diluted sera, and 94 % in 1:200 diluted sera. But the negative identical ratios between AGD and ELISA tests are 97 % and 99 % respectively in 1:100 and 1:200 diluted sera. The positive identical ratios between CIEP and ELISA tests are 98 % and 96 % respectively in 1:100 and 1:200 diluted sera, but also 97 % and 99 % in 1:100 and 1:200. Control sera, such as clonorchiasis, amoebiasis and toxoplasmosis, revealed all negatives with Paragonimus antigen in AGD, CIEP and ELISA tests. By above results, ELISA was most sensitive, next CIEP and AGD. But AGD test appears to be more useful when used to crude antigen without cross reaction with other parasitic infections. CIEP test is basically equal in terms of precipitin reaction, but CIEP is able to be detected more sensitively and rapidly though less simple in handiwork than AGD. Consequently, three methods for immunological tests of paragonimiasis have good correlations with one another. Also, each of these has both merits and demerits in immunological test for paragonimiasis. But the ELISA test was proved to be the most sensitive and convenient tool for mass screening test, especially in case of using purified antigen.

Two Different Anti-Galactan Lectins in Eel Serum

Two different anti-galactan lectins from eel serum (Anguilla anguilla) were isolated by affinity chromatography on fucogel and on 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine coupled to Aminohexyl-Sepharose. The fucolectin and the CRP-analogue protein thus purified directly from the eel serum were characterized by SDS-gradient gel electrophoresis, immunoelectrophoresis, and by agar gel diffusion tests. The CRP-analogue protein with a molecular weight of 24.800 Daltons was present in a high concentration. Carbohydrates are absent in both the proteins, showing that the fucolectin and the CRP analogue do not belong to the immunglobulin/antibody group of serum proteins.

Bound and spontaneously released toxic products of NAG vibrios cultivated in minimal synthetic media

Properties of toxic substances released spontaneously and by ultrasonic disintegration from NAG vibrio cells cultivated in minimal synthetic medium were compared.The released material was separated on the AMICON ultramembranes, dialysed and fractionated on the column of Sephadex G-100. This procedure revealed three fractions released by ultrasonic disintegration. The culture released spontaneously only one fraction separated by the above metioned procedure. The molecular weight estimated by the elution from calibrated G-100 column of the fractions released by the ultrasound was a follows: first fraction (H1) over 100,000, second fraction (H2) 25,000–30,000 and third one (H3) below 10,000. The molecular weight of the spontaneously released fraction (F1) was over 100,000. The biological activity of the fractions was tested by the rabbit skin test and by the rabbit ligated ileal loop test. The fractions H1, H2 and F1 were positive in both while the fraction H3 was negative. Fraction F1 was inactivated at 56 °C while at 100 °C its activity was restored. In agar gel diffusion test, the presence of endotoxin in fraction F1 was proved by means of specific antiserum. Results show that the toxic substances of enterotoxin nature are bound also to cell structures. They reveal the biological activity qualitatively and quantitatively different of the spontaneously released substances. Besides the endotoxin also other cell components can contribute to these effects.

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme.

Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trypsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.

Detection of Dermatophilus congolensis antibody in the milk of strcptothricosis infected cows

Passive haemagglutination and agar gel diffusion tests were used to detect specific antibody to Dermatophilus congolensis antigens in serum and milk of eight streptothricosis infected Friesian milking cows. All the sera and milk samples showed the presence of antibody but titres were higher in sera. Precipitating antibodies were detected only in three sera. A possible implication of this finding is discussed with respect to passive immunity in the young calves from infected dams.

Isolation of cowpox virus clones deficient in production of type A inclusions: relationship to the production of diffusible LS antigen.

Cowpox virus clones (A- clones) deficient in production of type A inclusions were isolated from two cowpox strains, Amsterdam and 53. These clones did not differ from their parents in major markers such as pock morphology in chorioallantoic membranes and pathogenicity in the rabbit skin. However, the LS antigens induced by A- clones developed precipitin lines in agar gel diffusion tests, while the antigens from their parents failed to precipitate. Immunofluorescence and agar gel diffusion tests revealed that antigens detectable by antisera against purified type A inclusions and LS antigens were closely related to each other. These findings suggest that the A- clones might be variants of cowpox virus which have lost the ability to assemble LS antigens into type A inclusions.

Isolation and identification of mycoplasmas from pig lungs in Singapore

A total of 656 lung specimens comprising 196 grossly pneumonic lungs from clinically diseased pigs and 230 grossly pneumonic and normal lungs from abattoirs were cultured for mycoplasmas. Mycoplasmas or acholeplasmas were recovered from 102 lung specimens, of which 28 were serologically identified (by disc growth inhibition and agar gel diffusion tests) as Mycoplasma suipneumonia, 14 as M hyorhinis, 35 as Acholeplasma granularum and 4 as A laidlawii. In addition, 15 isolates were unclassified and six were not typed. Mycoplasmas isolated from the suspect enzootic pneumonic lungs: slaughterhouse pneumonic lungs: normal lungs were in the ratio of 3:2:1. M suipneumonia, A granularum and A laidlawii were isolated from both normal and pneumonic lungs of pigs while M hyorhinis was only isolated from pneumonic ones. A close antigenic relationship between M suipneumonia and A granularum was demonstrated.

Antigen analysis of the polysaccharides of Xanthomonas campestris pv. oryzae.

The diffusible antigens of Xanthomonas campestris pv. oryzae were analyzed by using antisera prepared with the isolates Q7472 (serovar A), Q7502 (serovar B-I) and N5837 (serovar B-II). When these antisera were added to the slime polysaccharide and somatic polysaccharide of the isolates Q7472, Q7502 and N5837 in test tubes, precipitation of the antibody occurred showing different quantitative curves, suggesting that slime and somatic polysaccharides of these isolates were not identical serologically. In agar gel diffusion test, the suspensions of whole cells, slime polysaccharide and somatic polysaccharide of the isolate Q7472 produced a few precipitin bands with all antisera. Among these antigens, the antigen named “a” was specific for serovar A. It was extracted with distilled water, saline solution and 0.5 N trichloroacetic acid and formed thick precipitin band with both homologous and heterologous antisera in agar gel diffusion test. It was heat stable, precipitated with ethanol and not affeced by Pronase treatment. All antigens other than “a” were not specific for serovars.

Canine parvovirus infection in a group of beagles (author's transl)

An outbreak of canine parvovirus infection in a group of 26 beagles was described. The disease characterized by acute intestinal involvement was highly contagious but fatality was low (1/26). Transient leukopenia was recorded in some animals. All the convalescent sera were positive in agar gel diffusion test using feline panleukopenia virus antigen. Histopathological examination on fetal dog revealed regressive change associated with formation of amphophilic intranuclear inclusion bodies in the enterocytes of the Lieberkühn's crypt and regressive change in the lymphoid tissues. Pathologic picture exhibited strikingly resemblance with that of feline panleukopenia.

SEPARATION OF CLOSTRIDIUM BOTULINUM TYPE A DERIVATIVE TOXIN INTO TWO FRAGMENTS

Two fragments with molecular weights of 105,000 (fragment I) and 58,000 (fragment II) were separated chromatographically from each other after Clostridium botulinum type A derivative toxin adsorbed onto a QAE-Sephadex column was treated with dithiothreitol and urea. They were antigenic and formed crossing precipitin lines against anti-derivative toxin in agar gel diffusion tests. Upon removal of dithiothreitol and urea by dialysis, the two fragments reassembled to reconstruct the derivative toxin molecule.

Nachweis von Leptospiren-Antikörpern in Seren immunisierter Kaninchen mit Hilfe des indirekten Immunfluoreszenztestes

Antibodies of Leptospirae were demonstrated in the sera of immunized rabbits by the indirect immunofluorescence test, only after a suitable dilution of the "anti-rabbit-gamma-globulin fluorescin conjugated" was carried out in a cross reference titration ("chessboard method"). A distinct crossreaction was observed when anti-icterohaemorrhagiae - and anti-bratislava-immunsera reacted with several serotypes out of the "interrogans complex". But only a weak crossreaction was found when both sera reacted with the serotyp patoc from the "biflexa complex". The evaluation of the indirect immunofluorescence test was complicated by a streaky background of fluorescence. The agargeldiffusion tests (Ouchterolony) exhibited four, three and two precipitation bands according to the conbination of antigens and immunsera.

Studies of immunogenicity of Westphal lipopolysaccharides from Pasteurella multocida in mice

The immunogenicity of Westphal lipopolysaccharides (LPS) from 2 strains of Pasteurella multocida was tested in mice. When lipopolysaccharide from P. multocida strain P-1234 (bovine source) was inoculated into CF1 or C3 H HeJ mice, less than 20 per cent of the mice survived homologous challenge infection 4 weeks later. However, 90 per cent, or more, of the CF1 or C3 H HeJ mice survived homologous challenge infection after immunization with formolized P-1234 bacteria. Sera from the CF1 mice, but not C3H HeJ mice, formed precipitin lines against P-1234 LPS in an agar gel diffusion test. Hyperimmune sera from rabbits inoculated with formolized P-1234 bacteria passively protected more than 80 per cent of the CF1 mice against challenge infection; however, sera from rabbits inoculated with P-1234 LPS or LPS adsorbed to sheep red blood cells protected less than 20 per cent of the challenged mice. Absorption of rabbit anti-whole cell sera with LPS slightly reduced the capacity of serum to passively protect mice against challenge exposure.

Purification and immunobiological properties of R antigen and its relation to M protein of type 3 group A Streptococcus.

R protein was extracted from type 3 group A streptococci with hot (95 degrees C) HCl and was purified by ammonium sulfate precipitation followed by molecular-sieve and ion exchange chromatography. Although the R3 antigen was present in a heterogeneous population of proteins ranging from 78,000 to 100,000 daltons in size, we were able to separate an R-rich fraction that contained minimal amounts of heterogeneous proteins as indicated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The final yield of the purified R protein was approximately 15 mug (dry weight) per g (wet weight) of washed and sedimented streptococci. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular size of approximately 78,000 daltons. Amino acid analysis showed lysine, glutamic acid, alanine, and aspartic acid as the predominant amino acids. A detailed comparison of the purified R3 protein with type 3 M protein indicated a similarity in composition and order of frequency of amino acids. However, the R3 antigen was found to be distinctive from the M3 antigen in agar gel diffusion tests. In addition, R3 and M3 proteins behaved differently in opsonophagocytosis tests and opsonization inhibition tests. Thus, R3 and M3 proteins produced precipitin lines of nonidentity with an unabsorbed antiserum against whole type 3 streptococci: M3-specific antiserum, but not R3-specific antiserum, enhanced the phagocytosis of type 3 streptococci. Purified M3 but not R3 protein was capable of inhibiting the type-specific opsonization of type 3 streptococci. The physicochemical resemblance between M and R proteins in general suggests a common genetic origin. Perhaps R proteins are variant forms of M proteins from which the antiopsonic determinant has been deleted.

Teichoic acid antibody determination by agar-gel diffusion: effect of using dilute antigen preparations.

Because the reported frequency of teichoic acid precipitins in controls and various patient groups has varied considerably among laboratories, we studied the effect of using various concentrations of staphylococcal extracts in agar-gel diffusion tests for teichoic acid antibodies. Of 25 normal sera, only 1 was positive against an undiluted extract, but 4 were positive against a 16-fold-diluted extract. Of nine sera from patients with staphylococcal bacteremia, two were positive at a higher titer against the diluted extract. A false-positive serum against the undiluted extract had a twofold titer increase against the diluted extract. Because human immune serum globulin is generally used as a positive teichoic acid antibody control, the variability of five different lots was studied. Three lots ahd teichoic acid antibody titers of 1:4, whereas one each had titers of 1:2 and 1:8. Based on this study, we feel that staphylococcal extracts should not be diluted. If immune serum globulins are used to determine the adequacy of ultrasonic extracts, newly acquired globulin lots should be standarized against an ultrasonic extract of proven sensitivity and specificity.

The use of counter current immunoelectrophoresis in the diagnosis of contagious bovine pleuropneumonia

A counter current electrophoresis technique for the rapid detection of Mycoplasma mycoides subsp, Mycoides antigen in tissue from animals infected with contagious bovine pleuropneumonia is described. The test proved sixteen times more sensitive than the standard agar gel diffusion test, but more tests are required with defined reagents to confirm its specificity.

Immunological reactions of Rift Valley fever virus strains from East and West Africa

Three strains of Rift Valley fever virus, namely Nigerian (NIG), Smithburn's neurotropic (SNT), and Lunyo variant (LUN) were compared by complement fixation (CF), neutralisation (N), haemagglutination/haemagglutination-inhibition (HA/HI) and agar gel diffusion (AGD) tests. They showed reciprocal cross-reactivity in CF tests. In N tests, using immune sheep sera, there was reciprocal cross-neutralisation between the NIG and SNT strains, but not with the LUN strain, the antiserum of which neutralised both NIG and SNT antigens whereas the reverse was not the case. When hyperimmune mouse ascitic fluid was employed in N tests, there was cross-reactivity between the three strains. Both the NIG and SNT strains yielded haemagglutinins, but not the LUN strain. Furthermore, by the antibody absorption and AGD techniques, the NIG and SNT strains were found to be identical and distinct from the LUN variant strain. The techniques found most useful in distinguishing between the three strains were HA and AGD. Laboratory neuro-adaptation of the classical pantropic virus did not appear to affect its haemagglutination activity.

Serological study of trichloroacetic acid extracts of Bacteroides fragilis.

Immunodiffusion techniques were used on trichloroacetic acid extracts from 10 strains of Bacteroides fragilis in detecting precipitating antibodies against this species in immune rabbit sera. Species and even strain specificities were observed in these precipitin reactions. Multiple antigens were detected in the extracts from some strains, whereas only one precipitin band per extract developed during agar-gel diffusion tests of others. The antigen extracts were found to be both heat stable and resistant to hydrolysis by alpha-chymotrypsin. Four serological patterns were demonstrated in homologous and heterologous reactions with the B. fragilis. antigen-antibody systems used. The results showed that some strains were serologically distinct from others, indicating that the strains tested are of more than one serotype.

Isolation and identification of avian mygoplasmas in Singapore

Two hundred and forty batches of chickens with chronic respiratory syndrome were tested for mycoplasmas. One hundred and five batches (43.8%) were found to have mycoplasmosis. A total of 110 isolates of mycoplasma was cultured, of which nine isolates were identified as Mycoplasma gallisepticum, 48 avian sero-group D, 45 M. gallinarum, one M. iners and seven unclassified. 2. Identification of the mycoplasmas isolated was carried out by biochemical and serological tests (disc growth inhibition and agar gel diffusion tests). A modified agar gel diffusion test using solubilised antigens with sodium deoxycholate-glucose solution was developed for serotying of mycoplasmas.

Trichinosis: epidemiology, clinical features and diagnosis (author's transl)

After ingestion of wild boar meat 58 patients developed typical clinical signs and symptoms of trichinosis. All patients had marked blood eosinophilia. In 45% of the patients GOT and GPT and in 62% HBDH were moderately increased, while in 76% LDH and in 86% creatine-kinase showed abnormally high values. CK-MB was increased in three patients. 92% of muscle biopsies showed typical morphological changes in skeletal muscles. Latexagglutination, complement-fixation and agar-gel diffusion tests were not reliable. In most of the examined cases clinical symptoms and histological results correlated well with the indirect haemagglutination test, the microprecipitation test with living larvae of Trichinella spiralis and evidence of specific antibodies (IgG, IgM, IgE) by indirect immunofluorescence and the ELISA technique. 25 patients were treated with thiabendazole (Minzolum), 19 of them successfully.