Biomedical subjects
Publications and source records attributed to A C Ward.
Analysis of the NS and M genes of the archetype H6N5 influenza virus strain A/shearwater/Australia/1/72 shows it to be a typical example of the avian host reservoir, containing Old World/Eurasian internal proteins with divergent surface proteins, which is a potential source of new pandemic strains.
Domestic sheep were sighted at different times from 1991 to 1993 on four Nevada (USA) ranges occupied by bighorn sheep. Nasal and pharyngeal swab samples were collected from both sheep species and cultured to determine if any strains of Pasteurella spp. were shared on range conditions after contact of the two species. Pasteurella spp. were isolated from all 38 bighorn sheep and 16 of 17 domestic sheep included in this study. The isolates were characterized on the bases of species, biotype, serotype, biogroup, and restriction enzyme analyses (REA) as well as ribotyping of bacterial DNA. A P. haemolytica biotype 3, biogroup 11 isolate from a domestic sheep had biochemical, REA, and ribotype profiles which were identical to those of isolates from three bighorn sheep on the same range. None of the other isolates were found to be common to the two sheep species. Disease was not detected in any of the bighorn populations. However, bighorn sheep populations were extirpated on two ranges while increasing on the other two, including the range on which P. haemolytica biotype 3, biogroup 11 strain was isolated. Declining sheep numbers were not correlated with the presence of any one strain of Pasteurella spp from the sheep.
We examined effects of a multivalent Pasteurella haemolytica vaccine (serotypes A1, A2, T10) on humoral immune responses and P. haemolytica isolation rates in bighorn sheep (Ovis canadensis). Thirty captive bighorns, divided into groups of three on the basis of age, sex, and previous history of pneumonic pasteurellosis, received 0, 1, or 2 vaccine doses. Mild, transient lameness in most bighorns 1 day after initial vaccination was the only adverse effect observed. Oropharyngeal (> or = 75%) and nasal (< or = 50%) isolation rates for P. haemolytica did not differ among treatment groups. Ten of 36 distinguishable biogroup variants accounted for about 87% of the 464 P. haemolytica isolates from bighorns, but prevalences of specific biogroups were not affected by vaccination. Bighorns receiving 1 or 2 vaccine doses showed marked elevations in leukotoxin neutralizing antibody titers beginning 1 wk after vaccination. Agglutinating antibody titers to serotype A1 and A2 surface antigens were also elevated in vaccinated bighorns within 2 wk after vaccination; agglutinating antibody titers to serotype T10 surface antigens were relatively high in all three groups but appeared unaffected by vaccination. Vaccination 7 to 14 wk prior to parturition elevated leukotoxin neutralizing antibody titers in colostrum, but neither leukotoxin neutralizing nor serotype A1 surface antigen agglutinating antibody titers differed through 16 wk of age among lambs born to dams from different vaccine dose groups. Our data demonstrate that this multivalent P. haemolytica vaccine is safe and stimulates marked antibody responses in bighorn sheep. Further evaluation of this vaccine as a tool in preventing and managing pasteurellosis in bighorn sheep appears warranted.
The addition of cAMP inhibits G-CSF-mediated proliferation and suppresses pRB phosphorylation in NFS-60 cells. We show that the latter could be attributed to different effects of cAMP in these cells: (i) down-regulation of the levels of cyclins D2 and D3, and cdk4, and (ii) induction of the p27Kip1 inhibitor of cdk4.
The myeloid cell line, NFS-60, is dependent on granulocyte colony-stimulating factor (G-CSF) or interleukin-3 (IL-3) for survival and growth. Long-term G-CSF-dependent proliferation was found to be completely inhibited by interferon-gamma (IFN-gamma), cyclic AMP, and dimethylamiloride and partially inhibited by IFN-alpha and lipopolysaccharide. With the exception of IFN-gamma, these agents exhibited a corresponding pattern of inhibition of DNA synthesis in quiescent NFS-60 cells stimulated with G-CSF. IFN-gamma was only a weak inhibitor of DNA synthesis, suggesting that it may act at a later stage to block proliferation. The addition of G-CSF to NFS-60 cells resulted in phosphorylation of the retinoblastoma protein (pRB) and activation of E2F DNA binding activity. The inhibitors were found to suppress the phosphorylation of pRB, lead to the production of higher order E2F complexes, and suppress the expression of c-myc and proliferating cell nuclear antigen (PCNA) to an extent that correlated with their ability to block DNA synthesis. These findings are consistent with the notion that the ratio of free/bound E2F binding activity is critical in controlling cell cycle progression through G1 to S-phase in these cells.
There is evidence that the cellular responses to cytokines, such as granulocyte colony stimulating factor (G-CSF) and interferons, depend on prior activation of components of the JAK/STAT signalling pathway. We report here that the myeloid cell line NFS-60 shows aberrant JAK/STAT signalling yet elicits expected biological responses to G-CSF and interferons-alpha/beta and gamma. Instead of increased phosphorylation of JAK1 and JAK2 in response to G-CSF and interferon-gamma, and JAK1 and Tyk2 in response to interferon-alpha/beta, we observed only an increase of phosphorylation of Tyk2 in response to all of these cytokines in NFS-60 cells. The subset of STAT proteins being activated in response to these cytokines was unusual as well. G-CSF activated STAT3 and STAT5A, whereas interferons activated, in addition to STAT1 and STAT5 other, as yet unidentified, DNA binding proteins. However, NFS-60 cells show normal biological responses to these cytokines, such as proliferation in response to G-CSF, and reduction of proliferation, induction of an anti-viral response and induction of specific genes in response to interferons.
Infection of mouse brain with influenza A virus has provided a valuable model for investigating viral adaptation and virulence. These studies have indicated important roles for the neuraminidase (NA), matrix (M), non-structural (NS) and haemagglutinin (HA) genes of the virus in determining neurovirulence. For the NA, three changes close to the active site have been identified in the neurovirulent strains, which also display altered enzyme properties, including changes in specificity. In the M gene, two specific amino acid substitutions in the M1 protein have been observed, Ala41-->Val and Thr139-->Ala, which correlate with increased virulence for mouse. Such changes are likely to affect the pH-dependent association/dissociation of M1 with the viral ribonucleoprotein, as well as growth and virulence. The changes in the NS gene in the neurovirulent strains cause alterations in the mRNA secondary structure to mask the 3' splice site, and correlate with reduced splicing of the NS gene in these strains. Finally, the increased virulence of the HA gene occurs by at least three different mechanisms: loss of a glycosylation site, a change at the cleavage site, and a substitution which may increase the pH of fusion. These observations define a useful set of parameters with which to analyse epidemic virus strains that have been associated with elevated CNS symptoms in humans. In addition, the changes present in the neurovirulent influenza strains show interesting parallels to those in the neurovirulent derivatives of other viruses, suggesting different viruses utilise common strategies to permit replication in the brain.
From November 1991 through March 1992, nasal and pharyngeal swab samples were collected from 45 bison (Bison bison) from Yellowstone National Park, Montana (USA) and cultured for Pasteurella spp. Thirteen isolates of Pasteurella spp. were recovered from 10 (22%) of the animals. Ten isolates were from pharyngeal samples in contrast to three isolates from nasal samples. Pasteurella haemolytica (six biotype T, two biotype A, and two biotype 3) was the predominant Pasteurella species. Five biotype T isolates were serotype 4 and the sixth agglutinated in antisera 3, 4, and 10. Both biotype A isolates were untypable with antisera to recognized type strains. Pasteurella multocida was isolated from the pharyngeal samples of one animal. Two isolates could not be identified to species.
OBJECTIVE: To determine the scope of strain variations among Campylobacter spp associated with abortion in sheep. DESIGN: To examine Campylobacter spp isolated from cases of abortion for biochemical, antigenic, and genetic differences. SAMPLE POPULATION: 15 isolates of Campylobacter spp isolated from cases of abortion during a single lambing season. PROCEDURE: Isolates were examined, using biochemical tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins (OMP), and DNA restriction enzyme analysis (REA). RESULTS: Eight strain variants were detected among the 15 isolates. 14 of the isolates were C jejuni, 13 of which were biotype I and 1 biotype II, and the remaining isolate was identified as C fetus subsp fetus. Five sodium dodecyl sulfate-polyacrylamide gel electrophoresis OMP patterns had distinctive profiles for C fetus subsp fetus and C jejuni biogroup-II isolates and 3 variants within the C jejuni biogroup-I isolates. Examination of REA patterns of DNA from the 15 isolates digested with Cfo I indicated clear differences correlating with species and biogroups and 4 REA variants among biotype-I isolates. CONCLUSIONS: Marked antigenic and genetic heterogeneity of Campylobacter isolates were associated with ovine abortion within a defined geographic area. CLINICAL RELEVANCE: Representatives of differing OMP and REA profile groups should be considered for incorporation in vaccines to optimize protection in this region and possibly other geographic areas.
Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cerevisiae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two H(S/F)RIG motifs. Electroporation with a series of peptides containing these motifs caused structural defects in yeast that resulted in osmotic sensitivity. A protein with functions relating to the yeast cytoskeleton, Sac1p [Cleves, A. E., Novick, P.J. & Bankaitis, V.A. (1989) J. Cell Biol. 109, 2939-2950], shows sequence similarity to Vpr, and Vpr's effect in yeast may be to disrupt normal Sac1p functions. The Sac1p equivalent has not yet been described in mammalian cells, but in rhabdomyosarcoma and osteosarcoma cell lines Vpr also caused gross cell enlargement and replication arrest [Levy, D.N., Fernandes, L.S., Williams, W.V. & Weiner, D.B. (1993) Cell 72, 541-550]. We note that there is a correlation between the region containing the H(S/F)RIG motifs and the pathogenicity of primate lentiviruses and we suggest that the function of Vpr may be to bring about cell growth arrest and/or cytoskeletal changes as an early step in HIV-1 infection.
A simple, but stringent, three group model of bacterial interstrain identity (two cultures of the same strain of Escherichia coli) and difference (a culture of a serologically distinct strain) was used in multiple serial weekly subcultures for five weeks to demonstrate the effect of both growth-related (phenotypic) and machine-related variation on pyrolysis mass spectra. An aliquot of serum from a single sample was included in each pyrolysis batch to distinguish machine drift from culture drift. Conventional principal component (PC) canonical variate (CV) analysis was successful within each pyrolysis batch but the variations between batches precluded the use of data from more than one batch in successful PCCV analysis. In contrast, artificial neural networks (ANNs) trained with data from one batch could be successfully used to identify groups in data from non-contemporaneous pyrolysis batches. Although the ANN method will require validation in more complex settings than this simple model, it is a promising approach to the problem of batch constraint in pyrolysis mass spectrometry.
The matrix (M) gene of influenza virus has been implicated as a determinant of virulence for mouse brain and lung. Comparison of the M gene sequences of the mouse brain adapted variants A/NWS/33 and A/WSN/33 to their parent, A/WS/33, identified two specific amino acid substitutions in the M1 protein which correlated with virulence for mouse: Ala41-->Val and Thr139-->Ala.
Influenza NS2 protein was expressed in Saccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.
The influenza virus A/WS/33 has been adapted to mouse brain to produce two neurovirulent derivatives, A/NWS/33 (NWS) and A/WSN/33 (WSN), with the viral neuraminidase gene shown to be the major determinant of neurovirulence. The complete nucleotide sequence of the NA genes from each strain has been determined, which has allowed the identification of changes that have occurred during adaptation to mouse brain. Five changes are shared by the neurovirulent strains. Comparison to the known neuraminidase structure has identified four of these that may affect the active site of the enzyme. In addition, significant differences in the properties of the neuraminidase from the neurovirulent strains were observed relative to the parent strain. While no correlation was observed between neurovirulence and overall neuraminidase activity or preference for a particular N-substitution, the enzymes from both neurovirulent strains showed an increased preference for small substrates and those with 2-->3 linkages, and their activity was potentiated by Ca2+ ions.
The neurovirulent strain of influenza A virus, A/NWS/33, is able to infect a large range of cell types, including mouse brain cells, which are not infected by its parent, A/WS/33. This seems to be largely due to the hemagglutinin of A/NWS/33. The complete nucleotide sequence of the HA genes of both strains has been determined and a comparison revealed a number of changes. Analysis showed that the virulence capabilities of the NWS HA involve at least three different mechanisms: (a) loss of a glycosylation site; (b) a change at the cleavage site; and (c) a substitution in HA2, which may increase the pH of fusion.
The nonstructural (NS) gene has been identified as an accessory factor in determining neurovirulence for influenza A virus. The nucleotide sequence of the NS gene of the neurovirulent variant A/NWS/33 was determined and compared with its parent, A/WS/33. Alterations in the mRNA structure of the gene were observed that serve to mask the 3' splice site. Changes in this region were shown to correlate with reduced splicing of the NS gene in neurovirulent strains.
Thirty-six encoded isolates of Escherichia coli. 32 of which were of serotype O157, were examined by pyrolysis mass spectrometry (PyMS). Thirty-one of the serotype O157 isolates possessed the flagellar antigen H7 and produced Verocytotoxin (VT), the other isolate serotyped as H45 and was non-toxigenic. Eighteen of the VT-producing E. coli (VTEC) isolates were from sporadic disease in residents of the Northern Region. Standard principal component (PC) and canonical variate (CV) analysis of the data distinguished only the four non-O157 isolates from the remainder which were indistinguishable by this approach. A similarity matrix based on differences between individual CV means distinguished a further ten isolates. The matrix correctly clustered 2 pairs of isolates from siblings and 4 isolates from an affected family. A further 5 clusters of 3 or more isolates and 6 pairs of isolates were defined. These groupings proved to be homogenous for toxin phenotype but occasionally entrained isolates of dissimilar phage type. However, in general, PyMS-derived clustering of apparently sporadic isolates accorded with geographical locations as determined by postcode. PyMS, which is a quick and high volume capacity phenotypic technique, may be a useful addition to existing methods in the investigation of the epidemiology of sporadic VTEC disease.
Bacterial isolates (including 17 Haemophilus somnus isolates and an H. somnus-like isolate) from asymptomatic or diseased cattle and sheep, were evaluated for markers associated with virulence and host predilection. The isolates were separated into 6 distinct biovariants, 3 for sheep and 3 for cattle, based on reactions in a battery of 21 test media. Three bovine isolates associated with disease caused hemolysis of bovine blood. The rest of the isolates did not hemolyze either bovine or ovine erythrocytes. Protein profiles of all H. somnus isolates were similar with the exception of the major outer membrane proteins (MOMPs). The MOMPs of isolates associated with disease in cattle had a relative molecular weight of approximately 41 kDa compared with 33 kDa for the MOMPs of isolates from asymptomatic cattle. The MOMPs from sheep isolates were either slightly higher or lower than the 41 kDa MOMPs of bovine isolates. Major antigens detected by Western blotting were similar in all isolates except the H. somnus-like isolate. An immunodominant 40 kDa antigen was conserved in all H. somnus isolates. Antibodies to this antigen have previously been found to be protective in cattle and may also be protective for sheep. Marked differences between cattle and sheep isolates were revealed by use of restriction enzyme analysis, which separated the isolates into 12 ribotypes and 15 unique DNA profiles. Thus, cattle and sheep isolates in this collection had distinctive differences in biochemical reactions, MOMP profiles, and DNA analyses. Such differences have potential value for epidemiological studies and may also be used to evaluate host specificity of H. somnus isolates.