Cowpoxvirus infection in the Patagonian cavy (Dolichotis patagonum) emerging disease in an educational animal park the first reported case.

Generalized cowpox infection in the Patagonian cavy may represent a threat to the health of immunocompromised persons. We report the first case of cowpoxvirus infection in the Patagonian cavy in an educational animal park. The mara developed extensive pox lesions, shedding large amounts of viral particles. The ending of vaccination programmes against smallpox in the late 1970's may lead to an increase in susceptibility of humans to zoonotic poxviruses.

Studies on the efficacy of hyperbaric rendering procedures in inactivating bovine spongiform encephalopathy (BSE) and scrapie agents.

The efficacy of the procedures in use at the two rendering plants in the Netherlands was assessed on a laboratory-scale using procedures that simulated the pressure cooking part of the rendering process. A pool of bovine spongiform encephalopathy (BSE)-infected brainstem from the United Kingdom and a pool of scrapie-infected brainstem from Dutch sheep were used to spike the rendering materials. The mixtures were subjected to various time-temperature combinations of hyperbaric heat treatment related to the conditions used in Dutch rendering plants in the early 1990s, and to the combination of 20 minutes at 133 degrees C required by the EU Directive on rendering of 1996. The efficacy of the procedures in inactivating BSE or scrapie infectivity was measured by titrating the materials before and after heat treatment in inbred mice, by combined intracerebral and intraperitoneal inoculations at limiting dilutions. Two independent series of experiments were carried out. The design of the study allowed for minimum inactivations of up to 2.2 log (2.0 in the second series) to be measured in the diluted infective material and 3.1 log in the undiluted material. After 20 minutes at 133 degrees C there was a reduction of BSE infectivity of about 2.2 log in the first series (with some residual infectivity detected), and in the second series more than 2.0 log (with no residual infectivity detected). With undiluted brain material there was an inactivation of about 3.0 log (with some residual infectivity detected). With the same procedure, scrapie infectivity was reduced by more than 1.7 log in the first series and by more than 2.2 log in the second series. With undiluted brain material there was an inactivation of more than 3.1 log. In each case no residual scrapie infectivity was detected. The BSE agent consistently appeared to be more resistant to heat inactivation procedures than the scrapie agent, particularly at lower temperatures and shorter times.

The effects of different steam-sterilization programs on material properties of poly(L-lactide).

As-polymerized poly(L-lactide) test rods were sterilized by seven different specially designed computer-operated autoclaving programs. As a control, common hospital sterilization was performed. In all cases, the molecular weight decreased after sterilization. A short time high-temperature sterilization led to less molecular weight decrease than a low sterilization temperature cycle with a longer sterilization time. Regular hospital sterilization significantly reduced the elongation at break and also resulted in a decrease of 35% in tensile strength. The program causing minimal damage to the material properties was studied in detail. This program, with a sterilization period of 60 s and 129 degrees C, was effective for PLLA sterilization and also looks very promising for sterilization of other thermo- and moisture-labile polymers.

Characterization of fastidious adenovirus types 40 and 41 by DNA restriction enzyme analysis and by neutralizing monoclonal antibodies.

The DNA of 48 strains of adenovirus type 40 (Ad40) and of 128 strains of adenovirus type 41 (Ad41), isolated between 1971 and 1986 from various countries, was characterized by restriction enzyme analysis using nine and ten restriction endonucleases respectively. Five new DNA variants of Ad40 and 18 new DNA variants of Ad41 were detected. Most of the restriction sites which differed among the various DNA variants appeared to be distributed at random over the entire length of the viral genomes of the two serotypes. The number of restriction sites by which two DNA variants differed from each other was used as a measure of their relatedness. Several clusters of closely related DNA variants were observed for each of the two serotypes. The 35 DNA variants of Ad40 and Ad41 were used to test monoclonal antibody preparations for their range of reactivity in a neutralization assay. One monoclonal antibody (5-8), raised against Ad40 strain Dugan, showed type-specific neutralization of all 11 Ad40 DNA variants tested. Six monoclonal antibodies, raised against Ad41 strain Tak, neutralized different proportions of the variants of Ad41. Two of these preparations (1-21 and 3-19) neutralized all 24 Ad41 DNA variants, while a third (1-23) reacted with only 12 Ad41 variants. Three other monoclonal antibody preparations (3-10, 3-18, 7-14) reacted specifically with only 6 of these 12 variants. The patterns of reactivity with the monoclonal antibody preparations correlated with the presence or absence of a HindIII restriction site at 56 map units and of an EcoRI restriction site at 52 map units on the Ad41 DNA. This region of the adenovirus DNA codes for the hexon protein, which is known to contain the type-specific neutralizing antigenic determinants.

A mouse model to estimate the potency of the diphtheria component in combined vaccines.

A mouse model is presented to estimate the potency of the diphtheria component in combined vaccines. In addition, a simplified mouse test is suggested for routine potency control of diphtheria. It will be important to discuss this approach to replace the lethal challenge test in guinea pigs.

Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.

An in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization assay and ELISA-SPOT test, respectively. After booster immunization with polio vaccine, anti-poliovirus-neutralizing ASC were present in circulation. This response was measurable between 5 and 12 days after booster vaccination. At between 12 and 90 days, another subset of B cells was found in peripheral blood that only produced poliovirus-specific neutralizing antibody after in vitro antigenic stimulation. The in vitro virus-induced response required B cells, monocytes, and T4+ (T helper) cells, and was shown to result from de novo protein synthesis. The anti-poliovirus-neutralizing response in vitro could be dissected in a type-specific and intertypic cross-reactive response by using various antigen concentrations for in vitro stimulation. Evidence was obtained by absorption studies for the existence of intertypic cross-reactive neutralization-inducing epitopes.

Analysis of antigenic relationships among influenza virus strains using a taxonomic cluster procedure. Comparison of three kinds of antibody preparations.

Hemagglutination inhibiting (HI) monoclonal antibody preparations (MA) were raised against six influenza A (H3N2) strains from the period 1977-1982. Twenty-three hybridomas were selected and titrated in HI assays against these strains and against 18 influenza A (H3N2) viruses isolated in The Netherlands during the seasons 1981-1982 and 1982-1983. Similar HI tests were performed with conventional post-infection ferret antisera and with ferret antisera adsorbed with heterologous strains of influenza A (H3N2) virus. The resulting serological data were subjected to a computerized taxonomic cluster procedure based on the Euclidean distance between viruses. With respect to the degree of separation between clusters the unadsorbed ferret antisera were inferior to the adsorbed antisera whereas the MA were superior to both. Our results demonstrate that computer programs based on numerical taxonomy can be helpful in processing large numbers of serological data and that MA are indispensable in epidemiological and diagnostic influenza studies.

Strain differentiation of polioviruses with monoclonal antibodies.

Panels of monoclonal antibodies raised against different poliovirus type 1, 2 and 3 strains, were tested in a micro-neutralization test and in a micro-enzyme linked immunosorbent assay against a large number of poliovirus strains. The results were compared with those obtained with the classical system of serodifferentiation using strain specific cross-absorbed antisera. For this purpose a theoretical pattern fitting computer program was developed, in which each strain could be compared with all the other strains of which the serological data had been stored in the memory of the computer. The results obtained with the panels of monoclonal antibodies coincided well with those obtained with the cross-absorbed antisera. Especially for the identification of virus isolates related to the Sabin vaccine strains, these panels of monoclonal antibodies proved to be valuable tools.

Comparison of monoclonal antibodies with ferret sera for the characterization of influenza A (H3N2) virus strains in a computer system.

A panel of murine monoclonal antibodies was generated against five different influenza A (H3N2) virus strains, isolated between 1977 and 1980. Monoclonal antibodies with defined specificities in haemagglutination inhibition tests for the strains against which they had been raised were selected. Eventually 18 of these were chosen and tested against a large number of recent H3N2 isolates. The results were compared with those obtained with antisera from ferrets immunized with the five strains mentioned above and with similar ferret sera after adsorption with heterologous strains. The serological data obtained with these three panels of antibodies were subjected to a theoretical pattern fitting computer analysis. A good correlation was observed between the results obtained with these three systems. However, the unadsorbed ferret sera proved to be less discriminating whereas the monoclonal antibodies provided additional information which could not be obtained with the other two systems.

Induction of antigen-specific antibody response in human peripheral blood lymphocytes in vitro by a dog kidney cell vaccine against rabies virus (DKCV).

In the present report an in vitro method for obtaining a secondary human antibody response to a dog kidney cell vaccine against rabies virus (DKCV) is described. Cultures of peripheral blood mononuclear cells from normal rabies-immune and nonimmune donors were stimulated in vitro by DKCV. The production of virus-specific antibody in supernatant fluids was monitored by ELISA. Antibody was produced by lymphocytes from rabies-immune individuals, whereas those of nonimmune subjects consistently failed to produce anti-rabies antibodies after in vitro stimulation with DKCV. The generation of the anti-rabies virus antibody response of lymphocytes stimulated with DKCV was shown to be an antigen-dependent, as well as an antigen-specific process. Optimal antigen-specific responses were observed at relatively low concentrations of antigen (10(-1) to 10(-2) micrograms/culture). At increasing concentrations of antigen in culture (greater than 1 microgram/culture), the anti-rabies virus response was suppressed. Antibody produced upon stimulation was capable of neutralizing rabies virus. The response to rabies virus requires T cell help because lymphocytes depleted of SE rosetting cells did not respond to an antigenic stimulus. Studies in which the same individuals were followed over time showed a sequential development of circulating B cell subsets. The system may provide a model for the study of human B cell differentiation in vivo and in vitro and may be valuable for testing the potency of rabies vaccines in vitro.

Monoclonal antibodies to polioviruses. Comparison of intratypic strain differentiation of poliovirus type 1 using monoclonal antibodies versus cross-absorbed antisera.

A panel of 10 monoclonal antibodies raised to 3 different poliovirus type 1 strains was tested in a micro-enzyme-linked immunosorbent assay and in a micro-neutralization test against 87 poliovirus type 1 strains. The results, evaluated in a newly developed system for intratypic strain characterization, were compared with the results obtained with the classical sero-differentiation system by using a small number of strain-specific, cross-absorbed antisera. The new system not only uses results obtained with strain-specific antibody preparations, but also uses the information obtained with monoclonal antibodies reacting with less unique antigenic determinants. In a theoretical pattern fitting computer program, each virus strain could be compared with all the other strains for which serological data were stored in the memory of the computer. The results obtained with the new system coincided well with those obtained with the classical system: all except one of the strains classified as Sabin-like or intermediate in the classical system scored 'perfect fit' or 'related' with the Sabin 1 vaccine strain in the new system. Likewise, all virus isolates classified as Kuwait-like in the classical system scored 'perfect fit' or 'related' with the Dutch Kuwait-like isolate strain 78-9030.