Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens.

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.

Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV.

A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.

Adaptation of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the determination of virus-neutralizing antibodies using the virus-neutralization assay.

The tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] has been widely used for bioassays. Herein is described the use of the MTT dye with a virus-neutralization (VN) assay to titer infectious bursal disease virus (IBDV)-neutralizing antibodies. A standard VN assay using chicken embryo fibroblasts (CEFs) and IBDV was used for the assessment of IBDV-neutralizing antibodies. The percent of CEF killing due to IBDV was quantitated using MTT, and the absorbance (A) data were used to calculate the VN antibody titer. This method of calculation offers the expression of VN titer in terms of units of activity per unit of volume.

Action spectrum of antiviral factor from chicken sera.

Rous sarcoma virus infections of regressor line chickens stimulate the transient production of antiviral factors in the serum. Earlier the present authors reported that a viral neutralization factor (VNF) inactivated Rous sarcoma virus during a 3-h incubation. The VNF is likely to have a broad antiviral and antimicrobial spectrum because it is active against several unrelated pathogenic poultry viruses. The present study measured the activity of VNF against Newcastle disease virus, infectious bursal disease virus, and infectious bronchitis virus. The VNF is active in immunologically incompetent systems and must be preincubated with the virus in order to inhibit it. Based upon the current experiments, it is proposed that VNF is not an immunomodulator but directly inactivates the virus. The VNF agent appears to be one of a newly identified class of nonspecific antiviral agents produced in vivo in chickens in response to a viral infection.

Transfer of blood lymphocytes and macrophages between histocompatible progressor and regressor chickens infected with Rous sarcoma virus.

The development of histocompatible White Leghorn (progressor) and Arkansas Regression (regressor) chicken lines was described. When challenged with Rous sarcoma virus, progressor chickens developed fatal tumors while the regressor chickens eliminated the sarcoma. When sensitized histocompatible peritoneal macrophages and blood lymphocytes were transferred from regressor donors to progressor recipients, they both eradicated growing tumors. Histoincompatible cells were ineffective in inducing tumor remission. Within the two age groups tested, the sensitized blood lymphocytes and macrophages were only effective when transferred between age-matched donor and recipient chickens.

Stimulation of progressor and regressor chicken leukocytes with Con A, PHA-P, and Rous sarcoma tumor antigens.

Progressor (Pr) and regressor (R) chickens from the University of Arkansas lines were used as leukocyte donors. Peripheral blood leukocytes were stimulated with two mitogens, concanavalin A (Con A) and phytohemagglutinin-P (PHA-P). Both Pr and R leukocytes displayed the same Con A-stimulated dose dependency on blastogenesis. The regressor leukocytes were stimulated more than the progressor leukocytes at high concentrations of PHA-P. There did not appear to be any correlation between the response of individual chickens to mitogens and their ability to regress a Rous sarcoma. We concluded that the R-Rs-1 gene, which controls the animal's response to Rous sarcoma, and the Con A and PHA-P response genes are probably located at different loci in the chicken genome. Mitogen-induced blastogenesis does not appear to be a useful index to predict regression response in the chicken. Under our experimental conditions, regressor leukocytes showed a sensitization to sarcoma antigens, but the progressor leukocytes did not. The data suggest that the R-Rs-1 gene does not confer a generally enhanced immune status to the chicken but acts as an Ir response gene to augment the immune response to specific antigens.

Subsite mapping of enzymes. Double inhibition studies.

Our earlier subsite mapping studies led us to believe that ground state distortion occurred when a glucopyranoside ring filled the site which held the substrate monomer unit transferred to water during hydrolysis. We tested this hypothesis by performing double inhibitor studies on two amylases (EC 3.2.1.1) of bacterial origin. A general theory for multiple inhibition of this type is developed and applied to these two enzymes. Our data are consistent with the hypothesis that ground state strain occurs when substrates are bound to carbohydrates. An explanation is offered to account for the fact that monomers give strictly competitive inhibition patterns. The subsite model predicts that noncompetitive or mixed inhibition patterns can occur.

Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cell-mediated cytolysis and for heterogeneity of lytic rate.

The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used.

Multimolecular substrate reactions catalyzed by caabohydrases. Aspergillus oryzae alpha-amylase degradation of maltooligosaccharides.

Aspergillus oryzae alpha-amylase degrades maltooligosaccharides by other pathways besides simple glycosidic bond scission. The utilization of the alternate pathways increases with the concentration of substrate implicating a multimolecular substrate mechanism. Reducing-end labeled and uniformly labeled maltooligosaccharides were used to elucidate these alternate degradation mechanisms. Condensation followed by hydrolysis is not a significant pathway. Transglycosylation is concluded to occur, but no single transglycosylation mechanism can account for all of the experimental data for maltotriose degradation. Rather, a combination of transglycosylations must be invoked.

Model for carbohydrase action. Aspergillus oryzae alpha-amylase degradation of maltotriose.

Aspergillus oryzae alpha-amylase catalyzes degradation of oligosaccharides by a variety of pathways. We present here a quantitative study of the degradation of maltotriose by this amylase. Our results lead to a scheme involving multiple transglycosylation reactions and shifted binding due to simultaneous binding of two substrate molecules. The scheme is able to account for the diverse body of information collected for the enzyme. The effect of substrate concentration on the products of maltotriose degradation is correctly predicted over a 10(4)-fold concentration range, and the time course of maltotriose degradation is closely approximated by this scheme. The initial velocity data, which show deviation from Michaelis-Menton kinetics, are also consistent with the formulated scheme. The scheme is proposed as a general model of carbohydrase action.

Interpretation of 51Cr-release data: a kinetic analysis.

An analysis of 51Cr-release data from cell-mediated cytotoxicity assays, plotted in the form of effector cell dilution curves, is analyzed in detail in terms of a saturation kinetics model for effector-target interaction. The effect of nonimmune (bystander) cells in the effector population is particularly examined. The theoretical development of the saturation model predicts that, at low concentrations of target cells, increasing proportions of bystander cells should broaden the range of effector cell concentration required to achieve a given increment of target cell lysis, but that at high target cell concentration this effect should disappear. Experimental validation of this prediction is presented.

Repetitive attack by Aspergillus oryzae alpha amylase.

The action of Aspergillus oryzae alpha amylase on reducing-end, and uniformly radiolabeled maltotriose through maltodecaose has been studied. The enzyme is found to hydrolyze more than a single glycosidic bond during enzyme-substrate encounters. The extent of this repetitive attack is quantitated.

Characterization and serology of the matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni before and after degradation by an endogenous proteinase.

The intact matrix protein from a nuclear-polyhedrosis virus of the cabbage looper (Trichoplusia ni), isolated after inhibition of an endogenous serine-type proteinase, was further purified by molecular-sieve chromatography. The matrix protein was associated with carbohydrate moieties, and the carbohydrate content was determined for the two major peptides isolated after proteolysis by the endogenous proteinase. The association-dissociation interactions of the intact and proteinase-hydrolysed monomer units were characterized at high and low pH. At pH1.9, proteinase-degraded matrix protein dissociated into two different peptide fractions, FI and FII. Fraction FII, a single peptide of 9400 daltons, comprised one-third of the monomer unit of 28 000 daltons. At pH9.5, the degraded peptides were tightly associated in units equivalent to the intact monomer. These monomer equivalents associated to form a series of interconverting aggregates. The predominant aggregate sedimented at 11S and had a mol.wt greater than or equal to 200 000. Two non-cross-reacting antigens were present in the aggregate mixture. The presence of these two antigens does not reflect the presence of two different matrix proteins; rather, the expression of the antigens correlates with the degree of aggregation of the matrix protein.

Subsite mapping of enzymes. Depolymerase computer modelling.

We have developed a depolymerase computer model that uses a minimization routine. The model is designed so that, given experimental bond-cleavage frequencies for oligomeric substrates and experimental Michaelis parameters as a function of substrate chain length, the optimum subsite map is generated. The minimized sum of the weighted-squared residuals of the experimental and calculated data is used as a criterion of the goodness-of-fit for the optimized subsite map. The application of the minimization procedure to subsite mapping is explored through the use of simulated data. A procedure is developed whereby the minimization model can be used to determine the number of subsites in the enzymic binding region and to locate the position of the catalytic amino acids among these subsites. The degree of propagation of experimental variance into the subsite-binding energies is estimated. The question of whether hydrolytic rate coefficients are constant or a function of the number of filled subsites is examined.

Subsite mapping of enzymes. Application of the depolymerase computer model to two alpha-amylases.

In the preceding paper (Allen and Thoma, 1976) we developed a depolymerase computer model, which uses a minimization routine to establish a subsite map for a depolymerase. In the present paper we show how the model is applied to experimental data for two alpha-amylases. Michaelis parameters and bond-cleavage frequencies for substrates of chain lengths up to twelve glucosyl units have been reported for Bacillus amyloliquefaciens, and a subsite map has been proposed for this enzyme [Thoma et al. (1971) J. Biol. Chem. 246, 5621-5635]. By applying the computer model to the experimental data, we have arrived at a ten-subsite map. We find that a significant improvement in this map is achieved by allowing the hydrolytic rate coefficient to vary as a function of the number of occupied subsites comprising the enzyme-binding region. The bond-cleavage frequencies, the enzyme is found to have eight subsites. A partial subsite map is arrived at, but the entire binding region cannot be mapped because Michaelis parameters are complicated by transglycosylation reactions. The hydrolytic rate coefficients for this enzyme are not constant.

Models for depolymerizing enzymes: criteria for discrimination of models.

Several models for the action of alpha amylase have been proposed to account for the nonrandom distribution of oligosaccharides in the amylase digests of polysaccharides. The preferred-attack model attempts to account for the nonrandom distribution by assuming that the probability for bond cleavage depends upon the position of the bond in the chain. The repetitive, or multiple-attack, model suggests that the nonrandom distribution of oligosaccharides arises because an amylase can form a cage-like complex with a substrate and attack it several times during a single encounter. The multiple-enzyme or dual-site model suggests that the nonrandom yield of oligosaccharides arises from the combined action of exo- and endo-enzymes. The effects of pH, inhibitors, and substrate chain-length on enzyme action have been studied in several laboratories to determine which of the three action-patterns best describes the action of alpha amylase. The influence of these variables on product distributions or enzyme action-patterns are mathematically modeled in the Appendix. The experimental data on porcine-pancreatic alpha amylase are discussed in the light of the derivations.