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Biomedical subjects

A A Potter

Publications and source records attributed to A A Potter.

At least 37 records · Page 2Linked to original sources
PubMed1995

Tissue reaction and immunity in swine immunized with Actinobacillus pleuropneumoniae vaccines.

These studies were done to develop a subunit vaccine for swine that would protect against disease, but not create unacceptable tissue reactions at the immunization site. Swine were used to evaluate the local effects of subunit vaccines prepared from extracts of Actinobacillus pleuropneumoniae serotype 1 containing one of a wide variety of adjuvants. The antigen was an anionic fraction of a saline extract of A. pleuropneumoniae (ANEX). The adjuvants used were vegetable oils (peanut, sesame, canola, or corn oils, vitamin E, or Lipposyn II emulsion); mineral oil (Marcol-52) and other materials (aluminum hydroxide, polyethylene glycol, Quil-A, Amphigen, or Emulsigen-Plus). Two types of experiments were done. In the 1st set of experiments, pigs were given multiple simultaneous injections in different sites and euthanized on days 1, 3, 7, 14, 21, or 28. Tissues were examined for gross and histopathological lesions. In the 2nd set of experiments, 48 pigs were allocated to 6 groups and vaccinated twice with a vaccine containing ANEX antigen combined with one of various adjuvants. Antibody responses and protection from challenge were evaluated. Among the adjuvants that were tested, mineral oils induced protective immunity, although the mineral oil Marcol-52 resulted in severe tissue reactions. The vegetable oils induced little protective immunity, and some of them were quite irritating. The response to the other materials ranged from little irritation or protection induced by the vaccine containing aluminum hydroxide to effective protection without irritation after vaccination with ANEX/Amphigen or ANEX/Emulsigen-Plus combinations. In conclusion, swine were protected against disease by a subunit vaccine that did not create unacceptable tissue reaction at the immunization site.

Actinobacillus Infections↗
PubMed1994

Veterinary vaccines.

Vaccination of animals for the prevention of infectious diseases has been practised for a number of years with little change in product composition. Recent advances in molecular biology, pathogenesis and immunology have laid the groundwork for the development of a new generation of veterinary vaccines based on pure subunits as well as live vectored bacteria and viruses. Along with novel methods of antigen preparation, the use of new adjuvants and delivery systems will permit targeting of the appropriate immune response as well as offering flexibility in terms of vaccination protocols. These new technologies are also being applied to the development of vaccines to enhance animal productivity and to control reproduction.

Journal Article↗
PubMed1994

Regulation of major histocompatibility complex class II expression by Pasteurella haemolytica leukotoxin.

Many properties have been associated with Pasteurella haemolytica leukotoxin and other repeat-in-toxin toxins, including their cytotoxic activity on various cells of the lymphoid and nonlymphoid systems as well as their ability to modulate the immunological activity of lymphocytes and monocytes. In this study, we assessed the ability of P. haemolytica leukotoxin to affect the expression major histocompatibility complex (MHC) class II molecules on bovine peripheral monocytes. Peripheral blood mononuclear cells were isolated from P. haemolytica leukotoxin-seronegative calves and incubated with various concentrations of authentic leukotoxin as well as the recombinant lktA gene product (LktA). Expression of MHC class II antigen on cells was evaluated by flow cytometric methods. The results indicated that both a crude, authentic leukotoxin preparation and LktA were able to affect MHC class II expression by inducing a marked downregulation of MHC class II expression on bovine monocytes. However, when cells were activated with gamma interferon (IFN-gamma), LktA and Lkt had little or no detectable effect. By using a cell line which expresses MHC class II only after activation by INF-gamma, we were able to confirm the observation that LktA had no effect on the expression of MHC class II after IFN-gamma treatment. Leukotoxin affected the functional capacity of monocytes to present antigen, as demonstrated by the ability of LktA or authentic leukotoxin to totally inhibit a mixed lymphocyte culture from MHC-mismatched calves. Thus, leukotoxin was able to downregulate constitutive expression of MHC class II expression, and we propose that this is a novel way in which this molecule can affect the immune function of monocytes, playing an important role in bacterial pathogenesis and survival of organisms at the infection site.

Animals↗
PubMed1994

A carAB mutant of avian pathogenic Escherichia coli serogroup O2 is attenuated and effective as a live oral vaccine against colibacillosis in turkeys.

Colibacillosis is a serious and economically important disease of the respiratory tract of chickens and turkeys. The serogroups of Escherichia coli commonly associated with colibacillosis in poultry are O1, O2, and O78. Although previous attempts to develop a vaccine have not been very successful, vaccination is still considered the most effective way of controlling the disease. Therefore, our laboratory has been involved in the development of an attenuated live vaccine that will be effective in the prevention of colibacillosis. The carAB operon coding for carbamoyl-phosphate synthetase, an essential enzyme in arginine and pyrimidine metabolism, was selected for study. Generalized transduction was used to transfer a Tn10-generated mutation from a laboratory strain to virulent avian field isolates of E. coli. Molecular techniques were used to determine the point of Tn10 insertion within the carAB operon. The insertion mutants were then cured of the tetracycline resistance gene of the transposon to select for antibiotic-sensitive and stable carAB mutants. The degree of attenuation obtained by the mutation was determined in day-old chickens. Typically, when 100-fold the 50% lethal dose (for the wild type) was given, no more than 50% mortality in the day-old chickens was observed. The deletion mutant of serotype O2 was also found to be avirulent in turkeys rendered susceptible to infection with hemorrhagic enteritis virus A. Turkey poults vaccinated orally at 4 weeks old with either the wild-type E. coli EC317 strain or its carAB mutant EC751 were completely protected from infection following challenge with the homologous wild-type strain. Our data indicate that carAB mutants of virulent avian strains of E. coli will be effective and safe as live oral vaccines for prevention of colibacillosis in poultry.

Administration, Oral↗
PubMed1994

The occurrence of Haemophilus somnus in feedlot calves and its control by postarrival prophylactic mass medication.

Three field trials were conducted in a large commercial feedlot in Saskatchewan to determine the prevalence of Haemophilus somnus in calves and to evaluate the efficacy of prophylactic mass medication with long-acting oxytetracycline on day 17 (1990, n = 1336), day 11 (1991, n = 4372), or day 8 (1992, n = 5632) postarrival. Hemophilosis accounted for > 40% of the mortality in feedlot calves each year. Haemophilus somnus was cultured from the blood of one febrile calf on day 1 (0.1%, n = 895), but it was not cultured from nasal swabs on day 1 or day 11 (n = 881) or from blood samples on day 11 (n = 883). Similarly, it was not cultured from nasal swabs or blood samples from sick calves first treated for bovine respiratory disease (BRD) (n = 219). Serological titers to H. somnus increased (p < 0.05) in unvaccinated calves from day 1 (Geometric mean titer = 11,846) to day 96 (Geometric mean titer = 63,712), indicating natural infection following feedlot entry. Calves that relapsed twice with BRD or died from BRD +/- hemophilosis had significantly (p < 0.06) lower titers to H. somnus on days 1 and 96 than those that did not relapse twice or die. Postarrival mass medication with long-acting oxytetracycline did not reduce (p > 0.05) the risk of hemophilosis mortality. However, it reduced (p < 0.05) the risk of BRD treatment by 14% and the risk of BRD mortality by 71%. Additional epidemiological studies of H. somnus are needed so that we can develop strategic medication and vaccination programs to reduce losses from hemophilosis.

Animals↗
PubMed1993

Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1.

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping. One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis. It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A. pleuropneumoniae. This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA. Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A. pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains. Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 1 isolate.

Actinobacillus pleuropneumoniae↗
PubMed1993

Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus.

Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an apparent molecular weight of 40,000. The nucleotide sequence of the entire DNA insert was determined. Two open reading frames coding for polypeptides with calculated molecular weights of 21,893 and 30,721 were identified. The larger open reading frame encoded LppB, while the smaller reading frame encoded a nonseroreactive protein with a relative molecular mass of approximately 18 kDa. The 16 amino-terminal amino acids of the deduced LppB polypeptide showed strong sequence homology to the signal peptide of secreted bacterial proteins, and the sequence Leu-Ala-Ala-Cys at the putative cleavage site corresponds to the consensus cleavage sequence of bacterial lipoproteins. Synthesis of the mature LppB lipoprotein in H. somnus was inhibited by globomycin, a specific inhibitor of signal peptidase II. LppB was localized to the outer membrane of H. somnus.

Amino Acid Sequence↗
PubMed1993

Characterization of Escherichia coli isolated from cases of avian colibacillosis.

Forty-four western Canadian isolates of Escherichia coli associated with colibacillosis of turkeys and chickens were examined for serotype, antibiotic resistance, and production of aerobactin. The isolates belonged to fourteen O serogroups, with 39% of the strains being non-typeable. A high frequency of resistance to tetracycline, kanamycin, neomycin, cephalothin, streptomycin and erythromycin was observed. Most isolates produced aerobactin. Ten E. coli belonging to serogroups O1, O2 and O78 were also examined for pili production, hemagglutination, serum sensitivity, production of iron-regulated outer membrane proteins (IROMPS), and virulence. All isolates examined produced pili, exhibited mannose-sensitive hemagglutination of avian red blood cells and produced IROMPS under iron-restricted growth conditions. The five isolates of serogroup O1 and O2 were resistant to killing by turkey serum and were highly virulent. Only two of the five isolates of serogroup O78 were serum resistant. No correlation between serum resistance and virulence was observed in serogroup O78.

Animals↗
PubMed1992

Immunization of pigs against Actinobacillus pleuropneumoniae with two recombinant protein preparations.

Two Actinobacillus pleuropneumoniae serotype 7 antigens were expressed in Escherichia coli and tested for their protective efficacy in an experimental pig model. The antigens used were a fusion protein containing the carboxy-terminal 70% of the +/- 103 kDa cytolysin and a full length 60 kDa protein which has been shown previously to bind transferrin. Pigs were immunized twice with 25 micrograms of either or both preparations. All pigs developed a strong humoral immune response comparable to that induced by infection. Upon challenge with an A. pleuropneumoniae serotype 7 strain, all immunized groups were less affected by the disease and showed significantly lower mortality than the controls (p less than 0.1). Pigs receiving both antigens had a tendency to recover faster than the controls or animals which were vaccinated with only one antigen. Protection was serotype-specific since no cross-protection was detected following challenge with an A. pleuropneumoniae serotype 1 strain. A dose-response experiment using the single antigens at 200, 50 or 12.5 micrograms per dose showed no difference in protection among the groups.

Actinobacillus pleuropneumoniae↗
PubMed1992

Effect of Haemophilus somnus on phagocytosis and hydrogen peroxide production by bovine polymorphonuclear leukocytes.

The interactions between bovine polymorphonuclear leukocytes (PMNs) and the bacterium Haemophilus somnus are known to be complex. In this paper, we evaluated the effect of H. somnus on PMN function using a flow cytometric (FC) technique that simultaneously determined the extent of phagocytosis and hydrogen peroxide production by PMNs, as well as using conventional techniques, such as the nitroblue tetrazolium (NBT) and chemiluminescence assays, to analyse the PMN respiratory burst. Results from the FC and chemiluminescence assays demonstrated that in vitro exposure of PMNs to logarithmically growing H. somnus reduced the respiratory burst of PMNs obtained from healthy calves. However, this reduction was not detected by the NBT assay. A decrease in phagocytosis by PMNs could also be shown using the FC assay. In addition, PMNs from calves with acute Hemophilosis (i.e. exposed to H. somnus in vivo) showed reduced activity when compared to PMNs from healthy calves. These in vitro and in vivo observations indicate that the modulation of bovine PMN function by H. somnus may contribute significantly towards the pathogenesis of the disease.

Animals↗
PubMed1992

Molecular chimerization of Pasteurella haemolytica leukotoxin to interleukin-2: effects on cytokine and antigen function.

A chimeric recombinant protein composed of the lktA gene product from Pasteurella haemolytica fused to bovine interleukin-2 (IL-2) was made. The LKT-IL-2 chimera was compared with recombinant bovine IL-2 with regard to the ability to induce proliferative responses and LAK cell activity in bovine peripheral blood mononuclear cells in vitro. In both instances, chimerization had no effect on IL-2 activity. Similarly, the LKT component was unaffected in its ability to induce an effective immune response after immunization. The adjuvant properties of IL-2 have been established in a number of models, and this effect was tested by using the chimera. A multiple-injection protocol of LKT-IL-2 was compared with single-dose administration of LKT. The results obtained indicate that while there was no increase in specific antibody production, the IL-2 component of the chimera may be able to affect antigen-specific proliferation, as assessed by limiting-dilution analysis. Use of cytokine-antigen chimeras may provide a valuable antigen-adjuvant formulation that is simple to produce and purify and thus have economic advantages over conventional preparations. Furthermore, chimerization will also ensure that the adjuvant acts at the same site as the antigen, thus optimizing immunostimulatory activity.

Animals↗
PubMed1992

Molecular cloning, nucleotide sequence, and characterization of a 40,000-molecular-weight lipoprotein of Haemophilus somnus.

A gene of Haemophilus somnus encoding the major 40,000-molecular-weight antigen (LppA) was cloned on a 2-kb Sau3AI fragment. The nucleotide sequence of the entire DNA insert was determined. One open reading frame, encoding a 247-residue polypeptide with a calculated molecular weight of 27,072, was identified. This reading frame was confirmed by sequencing the fusion joint of two independent IppA::TnphoA gene fusions. The 21 amino-terminal amino acids of the deduced polypeptide showed strong sequence homology to the signal peptide of secreted proteins, and the sequence Leu-Leu-Ala-Ala-Cys at the putative cleavage site is identical to the consensus cleavage sequence of lipoproteins from gram-negative bacteria. The presence of the lipid moiety on the protein was shown by incorporation of radioactive palmitic acid into the natural H. somnus protein. Palmitic acid could also be incorporated into the recombinant protein in Escherichia coli. Synthesis of the mature LppA lipoprotein was inhibited by globomycin, showing that cleavage of the signal peptide is mediated by signal peptidase II in both organisms. By using site-directed mutagenesis, the cysteine residue at the cleavage site was changed to glycine. Radiolabelled palmitate was not incorporated into the mutated protein, showing that lipid modification occurs at the Cys-22 residue.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.

An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.

Actinobacillus pleuropneumoniae↗
PubMed1992

Characterization of two genes encoding distinct transferrin-binding proteins in different Actinobacillus pleuropneumoniae isolates.

The gene encoding the Actinobacillus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70% of the protein was expressed as an aggregate protein in Escherichia coli. The nucleotide sequences of the tfbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that of A. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The tfbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tfbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.

Actinobacillus pleuropneumoniae↗
PubMed1992

Cloning, sequencing, expression, and functional studies of a 15,000-molecular-weight Haemophilus somnus antigen similar to Escherichia coli ribosomal protein S9.

Haemophilus somnus is a gram-negative bacterium capable of causing a number of disease syndromes in cattle. This article describes the cloning and characterization of a gene coding for a 15,000-molecular-weight (15K) polypeptide which reacts strongly with antiserum against H. somnus. Analysis of plasmid-encoded polypeptides by polyacrylamide gel electrophoresis showed that the corresponding gene is the second in a transcriptional unit. The first gene codes for a protein with a molecular weight of approximately 17,000. Using antiserum against the two recombinant proteins, we could show that the natural proteins are predominantly present in purified ribosomes from H. somnus. The nucleotide sequence of both genes and flanking regions has been determined, and the deduced amino acid sequence of the two polypeptides was used to search for sequence homology in the GenBank data base. The 15K polypeptide showed 89% similarity to the Escherichia coli ribosomal protein S9, and the 17K polypeptide showed 94% similarity to the E. coli ribosomal protein L13. In E. coli, the corresponding genes constitute a bicistronic operon, with the same gene order as that found in H. somnus. A plasmid expressing the 15K protein was found to complement an E. coli rpsI mutation. When a frameshift mutation was introduced into the 15K protein gene, the resulting plasmid failed to complement this rpsI mutation, demonstrating functional homology between the 15K protein and S9 from E. coli. Downstream from the 15K protein gene is located another open reading frame, which could code for a polypeptide with a predicted molecular weight of 24,427. A protein with a similar molecular weight was detected in minicells containing the recombinant clone. This polypeptide is 69% similar to the stringent starvation protein (Ssp) of E. coli.

Amino Acid Sequence↗
PubMed1991

Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7.

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity with a frequency of approximately 10(-4). The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.

Actinobacillus pleuropneumoniae↗
PubMed1989

Identification of a maltose-inducible major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae.

The addition of maltose to the growth media of Actinobacillus pleuropneumonia (serotype 1) resulted in the induction of an outer membrane protein (OMP) with a molecular mass of 42 kDa. This protein had porin-like properties in that it was peptidoglycan-associated and was resistant to proteolysis by trypsin. A pleuropneumoniae expressing the 42 kDa OMP were unable to bind lambda phage. Similar proteins were also induced in A. pleuropneumoniae isolates representing serotypes 2 to 7 with the exception of serotype 4; however, not all isolates of any given serotype expressed a maltose-inducible OMP. Western immunoblotting using convalescent antiserae against the serotype 1 A. pleuropneumoniae indicated that the 42 kDa OMP was expressed in vivo and was cross-reactive with the maltose-inducible OMPs from other serotypes.

Actinobacillus↗