Wet and dry density of Bacillus anthracis and other Bacillus species.

AIMS: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1.162 and 1.165 g ml(-1) and the other group included strains whose spores showed higher density values between 1.174 and 1.186 g ml(-1). Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R(2) = 0.95) between density and volume for the spores of all strains and species studied. CONCLUSIONS: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.

Difference between the spore sizes of Bacillus anthracis and other Bacillus species.

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.

Phylogenetic analysis of bovine pestiviruses: testing the evolution of clinical symptoms.

This study presents a phylogenetic analysis of 115 bovine pestiviruses. A sequence data set from the 5' untranslated genomic region was analyzed with maximum parsimony, bootstrapping and parsimony jackknifing. We tested for the proposed classifications of the group and analyzed the evolution of the symptoms associated with Pestivirus infections in bovines. Based on the historical framework provided by our phylogenetic trees, we also characterized the extent and importance of contamination caused in biologicals by the virus. Our phylogenetic analyses showed that the previously defined genotypes are monophyletic, except for genotype 1a. Based on our cladograms, we propose the existence of more than 12 monophyletic groups within the species BVDV 1. The mapping of clinical symptoms suggests that the emergence of some genotypes could have been driven by a change in the pathogenic process. Enteric Problems appear to be ancestral, while Reproductive and Respiratory Problems arise with the emergence of genotypes 1b, 1d and the herein-proposed genotype Arg 1. The distribution of contaminant strains on the cladograms shows that pestiviral contamination is a common process, and also suggests that a contaminated product might be a vehicle for virus dispersion. Implications for virus evolution, virus taxonomy, veterinary medicine and biotechnology are discussed.

Measles virus circulation in Argentina: 1991-1999.

Nucleoprotein (N) and Haemagglutinin (H) genes from measles viruses isolated from Argentina before and after the 1993 and 1998 massive vaccination campaigns were characterised to determine genetic variations that occurred from 1991 to 1999. Measles viruses from the 1991-94 period were clustered with the C1 genotype and those from 1997-99 with D6. Genetic variations within the 1997-99 outbreak were less than 1.2% and 0.79% for the N and H sequences respectively. The C1 genotype has not been detected since 1994 and the finding that a single D6 virus was found in November 1999 demonstrates that wild type viruses are still circulating among a partially covered population.

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

Host soluble factors with blocking and stimulating activity on the expression of the bovine leukemia virus.

The bovine leukemia virus (BLV), like the human T cell leukemia viruses (HTLV-I and -II), is usually present in its host in a transcriptionally repressed state. However, the viral genome becomes derepressed a few hours after the infected lymphocytes are cultured in vitro. Depending upon the concentrations tested, plasma and lymphatic fluid of BLV-infected cattle have either stimulatory (BSF) or inhibitory (PBB) activity on viral expression in these cultures. These activities can be separated by chromatographic procedures. BSF is either an antiviral antibody or a BLV-induced molecule that binds to IgG. After complete removal of BSF, the PBB activity can be more consistently detected in bovine plasma and lymphatic fluid. PBB activity can also be demonstrated in human plasma. It seems likely that this activity is responsible for the latent state in which BLV, HTLV-I and -II, and human immunodeficiency virus are usually present in their hosts.

Induction and inhibition of bovine leukaemia virus expression in naturally infected cells.

Bovine leukaemia virus (BLV) resides in infected lymphocytes in a latent, repressed state but becomes expressed a few hours after the cells are cultured in vitro. We have identified several conditions and factors affecting the expression of BLV in short-term cultures of naturally infected lymphoid cells. The presence of foetal calf serum in the culture medium greatly stimulates virus expression. This stimulation is not due to cellular proliferation. Transcription of BLV RNA and synthesis of p25 in the cultures of peripheral blood lymphocytes are preceded by a lag period of several hours. Synthesis of BLV p25 in these cultures takes place almost immediately after viral RNA synthesis. Extending previous results, we demonstrate that the plasma and lymphatic fluid of cattle contain factors that suppress and stimulate BLV expression. As a result of systematic examination of several parameters, we have developed reproducible assays for the detection of these factors. It is very likely that their relative concentration in the host is an important determinant of susceptibility and resistance to the development of lymphosarcoma and persistent lymphocytosis in BLV-infected cattle.

The trans-activating C-type retroviruses share a distinct epitope(s) that induces antibodies in certain infected hosts.

Using sera from hosts infected with bovine leukaemia virus (BLV), human T cell lymphoma virus types I and II (HTLV-I and -II), or simian T cell lymphoma virus type I (STLV-I), we found that the major gag proteins of these viruses cross-react immunologically. The specificity of this cross-reactivity was demonstrated by absorption using purified viral proteins, virus lysates and extracts of infected cells. The data strongly suggested that the cross-reacting epitope(s), referred to as CE, differs from those responsible for cross-reactions between the major gag proteins of HTLV-I, HTLV-II and STLV-I, and between those of BLV and HTLV-I reported previously. The prevalence of antibodies to CE was low, even amongst infected hosts with high titres to other epitopes present in the major gag proteins of the homologous viruses. CE was not detected in any of the other C- or D-type retroviruses, or lentiviruses examined. Therefore, it is likely that CE can be used to define serologically a subgroup of C-type retroviruses, the genomes of which display unique features and functional activities.

Kinetics of inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole on calf thymus casein kinase II.

The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a specific inhibitor for RNA polymerase II transcription in vivo and in vitro [Tamm + Sehgal (1978) Adv. Virus Res. 22, 187-258; Zandomeni & Weinmann (1984) J. Biol. Chem. 259, 14804-14811]. The effect on RNA polymerase II-specific transcription seems to be mediated by its inhibition of nuclear casein kinase II [Zandomeni, Carrera-Zandomeni, Shugar & Weinmann (1986) J. Biol. Chem. 261, 3414-3419]. Inhibition studies indicated that DRB acted as a mixed-type inhibitor with respect to casein and as a competitive inhibitor with respect to the nucleotide phosphate donor substrates. The DRB inhibition constant is 7 microM for the calf thymus casein kinase II, with regard to both ATP and GTP.

The cap structure of simian hemorrhagic fever virion RNA.

The molecular weight of simian hemorrhagic fever virus RNA is 4.19 +/- 0.53 X 10(6) as determined by electron microscopy. Its base composition is 19.5 +/- 0.3 A, 33.3 +/- 0.3 U, 26.7 +/- 0.9 G, and 19.7 +/- 0.3 C per 100 nucleotides. The RNA of simian hemorrhagic fever virus contains a single type I cap per molecule, in the form m7G(5')ppp(5')Am.