The effects of gamma irradiation on some properties of two Aeromonas proteinases

The proteinases A and B of Aeromonas liquefaciens and proteinase B of Aeromonas salmonicida have, in the crude and purified state, been exposed to various doses of γ-irradiation from a Co⁶⁰ source, and the D₃₇ values indicate that the proteinase A is the most resistant.Casein was shown to have a marked protective effect on both proteinases during the irradiation, and while solutions of the purified enzymes were inactivated by doses usually used for food pasteurization, the crude enzymes, or solutions of purified enzymes to which casein was added, required doses usually used for food sterilization before being inactivated. Only minor effects of the environmental pH were observed. The antigenic properties of the enzymes seemed to be qualitatively unchanged in solutions exposed to 150 krad as observed using the casein precipitation inhibition test, and the irradiated proteinases were also inhibited by the naturally occurring proteinase inhibitors in the immune sera. The enzymoserological properties were not influenced by the changes in electrophoretic migration which were demonstrated by the zymogram technique. These proteinases are suitable as models for the examination of the physical properties of food spoiling enzymes and also for taxonomical work.Keyword: gammairradiation, preservation, Aeromonas, proteinases, enzymes, CP -test, zymograms     

Serodiagnosis of infections of pigs with Stephanurus dentatus

A serological survey of 135 pigs over 7 months old, some of which had liver and renal lesions in the presence ofStrephanurus dentatus, was undertaken for the presence of antibodies to S. dentatus. Only one sample gave a positive gel immunodiffusion reaction with crude whole worm extracts and none with immunoelectrophoresis. Oral inoculation of pigs with 1200 or 2000 L3 resulted in a specific reaction of anodal lines on immunoelectrophoresis from Day 37, and with double diffusion up to 4 specific lines were produced beginning from Day 43–53 using adult and juvenile antigens. Juvenile antigens gave more intense lines. Serological responses remained positive until 76–80 days post-infection. At necropsy infected pigs had fibrotic lesions in livers and portal thrombi containing larval S. dentatus even after 270 days. It is concluded that immunodiffusion and immunoelectrophoresis have limited usefulness in serodiagnosis of naturally occurring stephanuriasis.     

The effect of microbial and animal proteinases on peptide- and protein-lignosulphonic acid complexes in agar gel

It is shown that various microbial and animal proteinases, with the exception of pepsin, have a lytic effect on peptide- and protein-lignosulphonic acid complexes in agar gel, and that this effect is characteristically inhibited by specific antiproteinases and naturally occurring serum inhibitors. The activity of the enzymes is studied at various concentrations of peptides and proteins, and at various pH values in the agar. The lysis phenomenon is seen in relation to the precipitation theory of peptide- and protein-lignosulphonic acid complexes, and to biochemical reactions taking place in certain natural ecosystems. Activity caused by different microbial proteinases is demonstrated in agar plates at pH 3.6. The possibility of using the method for studies on proteolytic enzymes at low pH values is suggested.     

Saponin adjuvants. V. Precipitation of serum components by non-purified saponin adjuvants in agar gel diffusion.

The immunologically active adjuvant Quil A does not induce precipitating antibodies in rabbits. This was tested by immunodiffusion of serum samples taken after repeated injections of Quil A. Quil A does not react non-specifically with any of 6 different animal sera tested (rabbit, guinea pig, horse, sheep, cattle, and pig). Two commercially available saponins with known adjuvant activity (Saponin MT, E. Merck, and Saponin P 3, Food Industries) produced non-specific precipitation in double gel diffusion tests with all the sera, as did crude extracts of the South-American tree Quillaja saponaria Molina.}This phenomenon in relation to the local tissue damage caused by non-purified saponin preparations is discussed.     

Evaluation of pseudorabies viral antigens in the agar gel immunodiffusion test.

Procedures designed to extract pseudorabies viral (PRV) antigens from PRV-infected tissue cultures were investigated to determine whether differences in extraction method had an effect upon the final concentrated antigenic product. All four of the preparations made from PRV-infected tissue culture cells (trypsin extract and disrupted cells) or entire PRV-infected cultures (polysorbate 80 extract and (NH4)2SO4 precipitate) contained relatively large amounts of the same antigen, whereas cell-free PRV-infected tissue culture fluids did not contain significant amounts of this antigen. Specific antibody directed against this antigen was present in all PRV antisera tested. Two other antigens were observed in some of the preparations, but PRV antisera varied in their ability to precipitate with these antigens. Therefore, the number of precipitation lines observed in agar gel immunodiffusion between PRV preparations and PRV-positive antisera depended both upon the extraction method used to obtain the antigen and upon the specificity of the selected antiserum.     

Proteinase, lipase and amylase activities in the small intestine of pigs suffering from colienterotoxaemia.

The activities of proteinases, lipases, amylases and the activities of proteinase inhibitors, as well as the numbers of Escherichia coli in the contents from the small intestine were examined for pigs suffering from colienterotoxaemia and for healthy pigs. Enzyme activities were determined using an agar diffusion test.Compared with healthy animals the activities of proteinases and amylases in diseased animals were reduced while lipases showed increased activity. In pathologically changed contents showing large numbers of E. coli, proteinases could not be demonstrated; however, proteinase inhibitors were found in these contents. In healthy animals, proteinase inhibitors were not demonstrated in ingesta-con-taining contents.In diseased animals, E. coli were found in large numbers in all parts of the small intestine. In healthy animals, E. coli was demonstrated especially in the posterior part of the small intestine and regularly in small numbers.The possible influence of digestive enzymes, especially proteinases and their inhibitors, on enterotoxins from E. coli is discussed.     

The humoral immune response of rainbow trout (Salmo gairdneri) immunised by various regimes and preparations of Aeromonas salmonicida antigens

Antibody production in rainbow trout to extracellular antigens was investigated. The following antigen preparations and immunisation regimes were used: native extracellular products (ECP) in Freund's complete adjuvant (FCA), intraperitoneally (i.p.) with and without booster; formalinized ECP in FCA, i.p. with and without booster; washed, formalinized A. salmonicida cells in FCA, i.p., with booster; native ECP in saline, i.m., four weekly injections at two different doses, 45 micrograms and 6 micrograms each injection (after the protocol of Shieh, 1985). Using crossed normal rainbow trout serum, i.p., single injection (after the protocol of Sakai, 1985). Using crossed immunoelectrophoresis all antisera contained precipitating antibodies to three to five ECP components except that from fish immunised i.m. with 6 micrograms protein where antibodies were undetectable. In no case were specific antibodies to ECP protease or haemolysin detected. In a rabbit immunised with formalinized ECP in FCA under a similar regime to the rainbow trout, antibodies to at least 15 ECP components, including protease and haemolysin, were detected. The assumption of a specific immune response to the protease, at least in respect of antibody production, in recent reports of protection afforded by vaccines composed of either crude ECP or partially purified protease (Shieh, 1985) or partially purified protease inactivated by normal serum (Sakai, 1985) is not supported by the present findings.     

Studies on strains of Pasteurella haemolytica not typable by the indirect haemagglutination test

Thirty strains of Pasteurella haemolytica which were untypable by the indirect haemagglutination (IHA) test were examined serologically by rapid plate agglutination (RPA), agar gel diffusion (AGD), crossed immunoelectrophoresis (CIE) and counter current immunoelectrophoresis (CCIE) tests. Nine serogroups were identified by CCIE. Serogroup specificity, dependent on two antigens, was present in heated saline extracts of cells. Single representative strains from two serogroups were not pathogenic for specific pathogen-free lambs.     

Purification of larval Taenia solium antigens by gel filtration.

Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.     

鸡致病性埃希氏大肠杆菌不同菌株间同源性抗原的研究

通过兔制备了３株引起鸡败血症的埃希氏大肠杆菌（Ｅ．ｃｏｌｉ）高免血清,对分离自新疆不同地区的３０余株鸡致病性Ｅ．ｃｏｌｉ进行了玻板凝集试验和双向免疫扩散试验。结果证明,在琼扩试验中,不同的Ｅ．ｃｏｌｉ菌株间存在着同源性抗原成份,这种同源性抗原在绝大多数Ｅ．ｃｏｌｉ间都有数种之多。但是,这种相互间的同源抗原在玻板凝集试验中有时并不能表现,甚至有沉淀线出现的血清与抗原之间在玻板凝集试验中也不能检测出来。     

The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis

Hyperimmune sera were produced by serial inoculation of rabbits with Vero cell-adapted, sucrose gradient-purified Nigerian peste des petits ruminants virus (PPRV) isolate. Two antisera produced, neutralized the homologous PPRV but not the heterologous rinderpest Kabette "O" virus. The antisera gave strong precipitin lines with purified PPRV antigens and were used to detect PPRV and rinderpest virus antigens from ante-mortem secretions and post-mortem tissue homogenates from PPR and rinderpest virus infected goats and cattle by the agar gel precipitation tests (AGPT). The hyperimmune sera gave good titration curves with both purified Nigerian goat and the United Arab Emirate wildlife PPRV isolates in the indirect enzyme linked immunosorbent assay (ELISA). Results of indirect ELISA showed that although there were some cross reactions with the rinderpest, canine-distemper and measles viruses, at 1:100 dilution, the antisera would give a positive signal with only the homologous PPR virus.     

The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases

We performed a combination of proteinase assay, either in solution or immobilized in sodium dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In the quantitative assay, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was hydrolyzed by crude extracts of L2 (3.0+/-0.2 nmol h(-1)mg of protein(-1)) and L3 (7.7+/-0.1 nmol h(-1)mg of protein(-1)) and that both activities were partially inhibited by trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 15% and 3%, respectively. Also, we demonstrated that the Nalpha-p-Tosyl-l-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117+/-24 nmol h(-1)mg of protein(-1)) and L3 (111+/-10 nmol h(-1)mg of protein(-1)), suggesting a predominance of esterase activity in the crude larval preparation. Interestingly, the specific activity of serine-proteinases was totally inhibited by phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10% of this enzyme class activity was inhibited in the L2 crude extract. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. These findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae.     

Molecular cloning of two Haemaphysalis longicornis cathepsin L-like cysteine proteinase genes.

Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides which has several limitations. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes in eukaryotes such as development regulation and nutrition, thus they can be considered as good target antigens for a tick vaccine. In the present study we used primers designed based on the consensus amino acid motifs flanking the conserved active sites C25 and N175 present in all papain-like cysteine proteinases to amplify by polymerase chain reaction, sequence and characterize two Haemaphysalis longicornis tick cysteine proteinase genes. Based on the nucleotide and deduced amino acid sequences, both genes were identified as members of the cysteine proteinase gene family by presence in their sequences of consensus motifs flanking the conserved active sites C25, H150 and N175 that are present in all papain-like cysteine proteinases. Both genes are about 1.2 kb in size and show high sequence homology predominantly to invertebrate cathepsin L-like cysteine proteinases.     

Antigenic similarity between squamous cell carcinoma of horn (horn cancer) and normal bovine foetal tissues.

Normal bovine foetal (liver and skin) and horn cancer tissue antigens were examined using double diffusion agar gel precipitation and immuno-electrophoretic tests to detect any cross reactivity among them. Rabbit horn cancer antisera absorbed with normal bovine liver, skin and horn core epithelium antigens, when tested with foetal skin and liver (4 to 6 months of gestation), revealed the presence of 2 foetal antigens in horn cancer. Immuno-chemically 2 of the horn cancer antigens were found to be identical to the bovine foetal antigens.     

Peptide-lignosulphonic acid precipitation zones in agar gel. A direct micro quantitative procedure for the determination of peptide-precipitating lignosulphonic acids in aqueous solution

The effects of incubation time and incubation temperature on precipitation zones of peptide-lignosulphonic acid complexes in agar plates are studied in relation to the mathematical dependence of zone diameter as a function of the concentrations of lignosulphonic acids in serial 2-fold dilutions. Optimal conditions are given for obtaining regression lines with not significant second degree coefficients under defined conditions.A diffusion unit, proposed as a measure of peptide-precipitating lignosulphonic acids in aqueous solution, is defined. The possibility is discussed of using the described procedure as a micro quantitative method for the determination of peptide-precipitating lignosulphonic acids in aqueous solution.     

Characterization of a new fimbrial antigen present in Escherichia coli strains isolated from calves.

Thirteen Escherichia coli strains isolated from calves with diarrhoea, supposed to carry a common antigen were examined for their hemagglutinating activity and compared by bacterial agglutination, double diffusion in two dimensions and by crossed immunoelectrophoresis (CIE). Two of the strains were examined also in the electron microscope. Most of the strains agglutinated red blood cells of horse, ox, guinea pig and chicken, of which the agglutination of ox erythrocytes was mannose-resistant (MRHA). None of the strains agglutinated human erythrocytes. All strains with MRHA of ox red blood cells, regardless to their O:K:H antigens could be agglutinated in unabsorbed or absorbed antisera produced against cultures C1209 (020:K-:H9) and C1213 (09:K36:H-) when live cells as antigens were used. None of these sera agglutinated reference strains carrying K88, K99, 987P, F41 or FY (Att25) antigen respectively. By the double gel diffusion test and by CIE in extracts (60 degrees C) of the strains a common heat labile antigen, responsible for the MRHA of ox red blood cells was identified. Electron microscopy revealed that this common antigen was represented by thin, long, hair-like fimbriae on cells of E. coli C1213, and that specific homologous antibodies attached to these fimbriae.     

Diagnosis of Rhodococcus equi infection in foals by the agar gel diffusion test with protein antigen

A protein antigen that reacted in the agar gel diffusion (AGD) test and which had equi factor(s) activity, was partially purified from the culture supernatant of Rhodococcus equi by successive column chromatography on diethylaminoethyl cellulose and Sepharose 4B. Employing a standard foal serum, the concentration of this antigen was adjusted for the AGD test. Optimal dilutions of the antigen reacted in the AGD test with sera from foals naturally infected with serologically different R. equi. The antigen prepared was considered suitable for use in field surveys of R. equi infection. Accordingly, four groups of sera were tested: those from 18 foals diagnosed as being infected with R. equi, those from 54 control foals with culture-negative R. equi pneumonia, arthritis or cellulitis, those from 46 diseased foals suspected of having R. equi infection and those from 51 clinically normal foals. A positive precipitation reaction was observed with sera from 100% of the first group, 69.5% of the third group and 17.7% of the fourth group. A negative reaction was obtained with sera from 100% of the second group.     

Cross-reactions between crude antigens of larval Taenia solium (Cysticercus cellulosae) and other helminths of pigs

A crude antigen extract of larval Taenia solium was shown by immunodiffusion (ID) and immunoelectrophoresis (IEP) to cross-react with rabbit antisera against pig serum proteins and larval T. hydatigena, and by enzyme-linked immunosorbent assay (ELISA) with antisera against pig serum proteins, Fasciolopsis buski, larval T. hydatigena, hydatid cyst, Hymenolepis diminuta and Dipylidium caninum. Immunoblotting demonstrated that the crude antigens extract contained epitopes of pig serum proteins of 48 and 66 kDa. The crude extract also contained a subunit of antigen B (95 kDa) which was also found in T. hydatigena and H. diminuta. Immunoperoxidase and indirect immunofluorescence studies showed that cross-reacting antigens were distributed mainly on the tegument of T. solium.     

A comparison of total and specific immunoglobulin levels in healthy Atlantic salmon (Salmo salar L.) and in salmon naturally infected with Aeromonas salmonicida subsp. achromogenes.

Healthy Atlantic salmon and salmon with a history of chronic natural Aeromonas salmonicida subsp. achromogenes infection were compared with respect to total serum protein and the concentration and specificity of serum immunoglobulin. The immunoglobulin level was measured using competitive ELISA and the specific antibody activity against Aeromonas salmonicida subsp. achromogenes was measured using double sandwich ELISA. Significant elevation of serum protein and immunoglobulin concentration was observed in the infected salmon compared with the healthy fish. This was accompanied by weak anti-A. salmonicida activity in the infected fish which seemed to contribute to the raised immunoglobulin level to only a limited degree.     

Enzootic bovine leukosis: Preparation of an antigen for the agar gel immunodiffusion test and statistical comparison with other antigens

The preparation of an antigen for the agar gel immunodiffusion test prepared by ultrafiltration of cell culture supernatant (chronically infected fetal lamb kidney cells) is described. The antigen obtained was compared by the single radial immunodiffusion test with an antigen prepared by ammonium sulfate precipitation (30%) and the commercial antigens (Pitman Moore, Seromed). The positive serum was used either undiluted or diluted 1:16 to test for the internal (P 24) and the external antigens (Gp 60) respectively.The results obtained indicated that the antigen prepared by ultrafiltration appeared to be much superior to the others, and contained Gp 60 which detects the commonly found antibodies in bovine leukosis virus-infected cattle.