Biomedical subjects

A A Potter

Publications and source records attributed to A A Potter.

At least 19 recordsLinked to original sources

PubMed2001

Surface-expressed mig protein protects Streptococcus dysgalactiae against phagocytosis by bovine neutrophils.

The mig gene of Streptococcus dysgalactiae, a major bovine mastitis pathogen, encodes two plasma protein-binding receptors, alpha2-macroglobulin (alpha2-M) and immunoglobulin G (IgG). In this study, the mig gene from one S. dysgalactiae isolate was cloned and expressed in Escherichia coli. The IgG receptor region encoded by mig was conserved in 16 S. dysgalactiae strains. An isogenic mig mutant was constructed by allele replacement mutagenesis of the wild-type gene in S. dysgalactiae. The IgG-binding activity was lost in the mig mutant strain, whereas the alpha2-M receptor activity was still expressed but was detected only in the culture supernatant. In flow cytometry phagocytosis and bacterial-colony-counting bactericidal assays, the wild-type strain was found to be significantly more resistant to phagocytosis and killing by bovine neutrophils (PMNs) than the mig mutant strain when bacteria were preincubated with bovine serum. We therefore speculate that the Mig protein of S. dysgalactiae plays a role in virulence of the bacteria by binding to the plasma protein alpha2-M or IgG and thus preventing phagocytosis by bovine PMNs.

Animals↗

PubMed2001

New approaches for antigen discovery, production and delivery: vaccines for veterinary and human use.

Vaccination of individuals has been practiced for many years and has been one of the most effective methods of controlling infectious diseases. Unfortunately, even with this success, society continues to suffer multi-billion dollar economic losses annually due to infectious diseases. These losses occur in all animal species as well as in humans. In order to further reduce these losses, academicians and companies are employing the multidisciplinary approach to develop better and safer vaccines. These include capitalizing on advances in molecular biology, chemistry, pharmacy, immunology, genomics, proteomics, and fermentation. Thus, we are moving from a more empirical approach to vaccine production to a more focused, and, hopefully, more logical approach to identification and production of protective antigens. Furthermore, formulation and delivery of these antigens in playing a major role in revolutionizing how we deliver vaccines to induce the most appropriate immune response and ensure protection. The current review summarizes some of these advances and speculates as to how future vaccines will be produced and delivered for the benefit of society.

Animals↗

PubMed2001

Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions.

The objective of this study was to characterize virulence factors of Escherichia coli isolates from broilers with simultaneous occurrence of cellulitis and other colibacillosis lesions. Thirty flocks were sampled and 237 birds with cellulitis were examined. Eighty-two (34.6%) of 237 birds condemned for cellulitis had gross lesions in the heart, air sacs, joints, or liver. In 58 chickens, E. coli was isolated from both the cellulitis and other lesions of colibacillosis, and 18.9% of the E. coli isolates from the 2 types of lesions belonged to the same O group. Escherichia coli of serogroups O78, O1, and O2 predominated. Isolates of the same serogroup that were derived from different lesions in the same birds had similar patterns of biotype, aerobactin production, serum sensitivity profile, antibiotic sensitivity, and K1 capsule production. Escherichia coli derived from cellulitis lesions produced virulence factors similar to those found in E. coli isolated from other colibacillosis lesions in poultry.

Animals↗

PubMed2001

Cloning and characterization of the gene coding for NADPH-sulfite reductase hemoprotein from Actinobacillus pleuropneumoniae and use of the protein product as a vaccine.

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate and screened with serum produced in pigs that had been vaccinated with the anionic fraction of a sodium chloride extract. One E. coli transformant was isolated that produced a large amount of a protein with an electrophoretic mobility of about 67,000 molecular mass. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by restriction enzyme digestion and nucleotide sequence analysis which showed strong homology with the cysI gene of E. coli. One open reading frame of 1764 bases in length was detected which encoded a cysI protein from serotype 1, with a calculated molecular mass of 66,678. The DNA encoding the protein was labeled with radio-isotope and the homologous gene was isolated from an A. pleuropneumoniae serotype 5a library. The serotype 5a gene was the same length, but the cysI protein from serotype 5a was slightly larger (66,849) due to 8 substitutions in the amino acid sequence. Expression plasmids containing cysI from either serotype of A. pleuropneumoniae complemented an E. coli cysI mutant. Pigs vaccinated with the recombinant cysI were protected from challenge with A. pleuropneumoniae of the homologous serotype.

Actinobacillus Infections↗

PubMed2000

Studies on cellulitis and other disease syndromes caused by Escherichia coli in broilers in Sri Lanka.

Cellulitis caused by Escherichia coli in broilers results in substantial losses to the broiler industry in North America and Europe due to condemnations at slaughter. The objective of this study was to identify cellulitis in broilers in Sri Lanka and to characterize the E. coli from cellulitis and other colibacillosis lesions. Twenty-four farms from the low- and mid-country were selected and bacterial isolations were obtained from 241 birds. Two hundred and ninety-one gross lesions were observed in these 241 birds and 162 E. coli isolates were obtained. Cellulitis was observed in 21% of the birds. Twenty-one per cent of the birds had multiple lesions due to E. coli. The frequency of detection of other disease syndromes was 162 (67%) birds with pericarditis, 26 (11%) airsacculitis, 24 (10%) hepatitis, 12 (5%) perihepatitis, and 16 (7%) polyserositis (a combination of pericarditis, perihepatitis and airsacculitis). Serogroups O78, O2, O85 and O88 were distributed among the 32% of typable E. coli and 81% of isolates were assigned to three biotypes. Forty-four per cent of the E. coli isolates produced aerobactin and 88% demonstrated resistance to the bactericidal effect of normal chicken serum. The majority of the E. coli isolates were resistant to the antibiotics commonly used in poultry. All the E. coli isolates were non-haemolytic and 25% of the isolates produced K1 capsule. This study demonstrated the presence of cellulitis in Sri Lanka and this report describes some of the phenotypic characteristics of the E. coli isolates.

Animals↗

PubMed1999

Protective capacity of the Pasteurella haemolytica transferrin-binding proteins TbpA and TbpB in cattle.

The transferrin-binding proteins TbpA and TbpB from Pasteurella haemolytica biotype A serotype 1 were tested for their ability to confer protection against experimental P. haemolytica infection when administered to calves in vaccine formulations containing one or both antigens. Vaccine groups included TbpB (single immunization), TbpB (two immunizations), TbpA, TbpA+TbpB and a placebo. All animals that received TbpB had measurable antibody titres against the antigen at the time of challenge, while those that received TbpA did not show an antibody response. The TbpA+TbpB group showed the best protection against experimental challenge. Protection correlated with anti-TbpB antibody levels. The enhanced protection in the TbpA+TbpB group suggests TbpA contributed to protection through the induction of a non-antibody-mediated immune response. Sera from the TbpB-immunized animals was cross-reactive with TbpBs from other P. haemolytica serotypes.

Administration, Cutaneous↗

PubMed1999

Pathogenesis of porcine Actinobacillus pleuropneumonia, part II: roles of proinflammatory cytokines.

The in vitro production of proinflammatory cytokines after stimulation with Actinobacillus pleuropneumoniae and the relation of these cytokines in vivo with the disease caused by A. pleuropneumoniae were investigated. Within 24 h, in vitro stimulation by A. pleuropneumoniae (serotype 1) preparations, including killed bacteria, bacterial culture supernatant, lipopolysaccharide, and bacterial extracts, porcine pulmonary alveolar macrophages (PAM) produced significant (P < 0.05) amounts of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) as measured by bioassays. The supernatants containing interleukin-8 from PAM after stimulation by bacterial preparations showed significant neutrophil chemotaxis, while bacterial preparations alone did not. After in vivo infection with A. pleuropneumoniae, the mean levels of TNF-alpha and IL-1 in serum, as measured by bioassays, were elevated 37- to 27836-fold for TNF-alpha and 11- to 5941-fold higher for IL-1 within 4 d post-infection, depending on the treatments, and remained elevated up to Day 7. Both cytokines were also detected in porcine lungs by bioassays and immunocytochemistry. The results indicated that both secreted and surface components of A. pleuropneumoniae can stimulate PAM to produce proinflammatory mediators. Neutrophil chemoattractants rather than bacterial components are the major factor causing acute lung inflammation. The elevation of TNF-alpha and IL-1 in pigs occurred coincident with the onset of acute clinical disease.

Actinobacillus Infections↗

PubMed1998

Intracellular survival of Haemophilus somnus in bovine blood monocytes and alveolar macrophages.

The mechanisms used by Haemophilus somnus to survive and multiply within bovine mononuclear phagocytes are not fully understood. In order to study the interaction between bovine mononuclear phagocytes and H. somnus, a colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenylItetrazolium bromide (MTT) was developed to assess the survival of H. somnus within cultured bovine blood monocytes (BBM). Using this system, it was found that H. somnus was able to survive within BMM in vitro, and the kinetics of its survival were similar to that seen in BBM isolated from experimentally infected cattle. Using ultrastructural studies, it was possible to demonstrate the survival of H. somnus in freshly isolated bovine mononuclear phagocytes in membrane-bound vacuoles. To determine if activation of macrophage function would result in elimination of intracellular H. somnus, BBM were treated with E. coli lipopolysaccharide (LPS) or recombinant bovine (rBo) cytokines, interferon-gamma (IFN-gamma), granulocyte macrophage colony stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). Treatment of BBM with rBoIFN-gamma, rBoGM-CSF or E. coli LPS resulted in decreased intracellular survival of H. somnus at 18 and 48 h, whereas BBM treated with rBoTNF-alpha or rBoIL-1beta had reduced intracellular survival of H. somnus only at 18 h. However, none of these treatments resulted in complete elimination of the intracellular bacteria. The ability of H. somnus to survive and multiply in both freshly isolated and cytokine-treated cultured BBM demonstrated the capability of H. somnus to escape from macrophage killing mechanisms. This capability may play a role in the dissemination of H. somnus infection in the body.

Animals↗

PubMed1998

The abp locus of Streptococcus uberis encodes a protein homologous to polar amino acid and opine binding proteins of gram-negative bacteria.

A gene locus abp was identified immediately upstream of the CAMP factor gene cfu in Streptococcus uberis. An open reading frame capable of coding for a 277-residue protein was identified. On the basis of sequence characteristics, the abp gene product is potentially a polar amino acid and opine binding component of an ATP-binding cassette type (ABC-type) transport system similar to those of Gram-negative bacteria. This membrane protein is likely lipid modified at its amino terminus and was present in five S. uberis strains and one Streptococcus parauberis strain examined.

ATP-Binding Cassette Transporters↗

PubMed1998

Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions.

Actinobacillus Infections↗

PubMed1997

Modulation of phagocytic function of bovine mononuclear phagocytes by Haemophilus somnus.

The interactions between bovine mononuclear cells and Haemophilus somnus are known to be complex. To study this interaction, a flow cytometric assay was developed to assess the effect of H. somnus on phagocytosis of killed opsonized Staphylococcus aureus by bovine alveolar macrophages and blood monocytes. Using this in vitro system, it was found that log phase H. somnus significantly inhibited the phagocytosis of killed opsonized S. aureus by bovine alveolar macrophages obtained both from healthy calves and from cattle experimentally infected with H. somnus. However, killed log-phase H. somnus, in vitro passaged and stationary phase H. somnus had no effect on the phagocytic activity of these cells. In contrast to bovine alveolar macrophages, blood monocytes showed a significant increase in their phagocytic activity following in vitro exposure to either log or stationary phase H. somnus. Using a lypophilic, non-toxic fluorophore PKH2 to label live H. somnus, it was possible to simultaneously measure the uptake of both S. aureus and H. somnus. Stationary and log phase H. somnus were taken up by macrophages equally well, even though phagocytosis of S. aureus was inhibited by only log phase H. somnus. These results demonstrate the ability of H. somnus to modulate bovine mononuclear phagocytic function which might contribute towards the pathogenesis of bovine hemophilosis.

Animals↗

PubMed1997

Effect of Haemophilus somnus on nitric oxide production and chemiluminescence response of bovine blood monocytes and alveolar macrophages.

Haemophilus somnus is able to survive and multiply in bovine blood monocytes (BBM) and alveolar macrophages (BAM), but the mechanisms used by H. somnus to evade killing mechanisms of bovine mononuclear phagocytes are not completely understood. To study the bactericidal ability of bovine mononuclear phagocytes following interaction with H. somnus, in vitro assay systems were developed to detect the luminol-dependent chemiluminescence response (LDCL) and nitric oxide (NO) production of BBM and BAM. Live logarithmically growing or stationary phase H. somnus inhibited the LDCL of BBM and BAM costimulated with opsonized Staphylococcus aureus. Inhibition of the LDCL response of BBM and BAM was not mediated by live H. somnus opsonized with hyperimmune serum, or by killed bacteria. H. somnus stimulated both BBM and BAM to produce NO at levels comparable with Escherichia coli lipopolysaccharide. While NO was being produced, viable H. somnus could still be isolated from the cell cultures. The ability of H. somnus to inhibit LDCL of both BBM and BAM, and resistance to NO killing may be an important mechanism that contributes to survival of the organism following ingestion by bovine mononuclear phagocytes.

Animals↗

PubMed1997

Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study.

Amino Acid Sequence↗

PubMed1997

Isolation of Escherichia coli from cellulitis and other lesions of the same bird in broilers at slaughter.

Cellulitis results in substantial losses to the broiler industry due to condemnations at slaughter. This study was conducted to clarify the association between Escherichia coli isolated from cellulitis and other lesions caused by E. coli in individual birds. Fourteen flocks were sampled and 118 birds with cellulitis were examined. Escherichia coli was isolated from all but 2 of the cellulitis lesions, and serogroups O78, O1, and O2 predominated. Thirty-six birds had at least 1 other lesion in addition to the cellulitis lesion. Isolation of E. coli from cellulitis and other lesions occurred in 7 of the 14 flocks. Escherichia coli of the same serogroup were isolated from cellulitis and other lesions in some birds, suggesting that a single E. coli may sometimes be responsible for both types of lesions.

Animals↗

PubMed1996

Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis.

The gene coding for the CAMP factor from a strain of Streptococcus uberis (ATCC 9927) was cloned in Escherichia coli. Chromosomal DNA from Streptococcus uberis was used to construct a gene library in plasmid pTZ18R and six CAMP-reaction positive clones were obtained from a total of 10,000 transformants. One clone, pJLD21, was subcloned and the CAMP factor gene was located in a 3.2 kb BamHI fragment. The nucleotide sequence of Streptococcus uberis CAMP factor gene was determined and the deduced amino acid sequence is highly homologous to the corresponding Streptococcus agalactiae protein. Immunoblot analysis revealed that the recombinant strain pJLD21 expressed a protein with a molecular weight of 28 000. Antibodies raised against purified Streptococcus uberis CAMP factor cross-reacted with Streptococcus agalactiae protein B.

Amino Acid Sequence↗

PubMed1996

Escherichia coli cellulitis in broiler chickens: clonal relationships among strains and analysis of virulence-associated factors of isolates from diseased birds.

Thirty-nine Escherichia coli isolates from broiler chickens with cellulitis were serotyped and analyzed for clonal relationships by multilocus enzyme electrophoresis. The isolates were further characterized with respect to hemagglutination (HA); serum resistance; antibiotic susceptibility; production of aerobactin, colicin V, and hemolysin; expression of K1 or K5 capsule; sensitivity to cloacin DF13 after treatment with diphenylamine; expression of iron-regulated outer membrane proteins; and virulence in 1-day-old chickens. In addition, the isolates were examined for the presence of DNA sequences related to F1A (fim) and P (pap) fimbriae, aerobactin synthesis (iuc) and transport (iut), hemolysin operon hly, and TraT lipoprotein-induced serum resistance (traT). Only 38.4% of the isolates were typeable with standard O antisera, and of these, serogroups O25 and O78 were the most frequently observed. Multilocus enzyme electrophoresis, based on 20 enzymes, resolved 17 electrophoretic types, forming seven clusters. Isolates from four of these clusters fell into E. coli clone complexes that have been previously reported to be commonly associated with avian colibacillosis. All isolates expressed two to five iron-regulated outer membrane proteins, were resistant to serum and cloacin DF13, and possessed DNA sequences homologous to fim and iuc/iut. Most isolates (72%) were positive for traT, and a majority produced colicin V and aerobactin (92 and 82%, respectively). Assays for the presence of fim and pap DNA sequences, for HA, and for virulence gave variable results but suggest that cellulitis isolates may express F1A and/or other mannose-resistant HA fimbriae different from P and may be virulent in 1-day-old chickens. Our results support the hypothesis that cellulitis in broilers in many cases is caused by E. coli clones identical to other pathogenic avian E. coli strains. Certain clones may be specific to cellulitis, because 25% of the isolates tested belong to clusters not related to known clone complexes.

Animals↗

PubMed1995

Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5.

The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A.A. Potter, and P.J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate.

Actinobacillus Infections↗

PubMed1995

Effects of various vaccination protocols on passive and active immunity to Pasteurella haemolytica and Haemophilus somnus in beef calves.

Two field trials were conducted in a beef cow herd in Saskatchewan to determine the effectiveness of a combined Pasteurella haemolytica and Haemophilus somnus vaccine in increasing passively and actively acquired antibodies in beef calves. Vaccination of dams at 4 and/or 7 weeks prepartum was associated with increased antibody titers to P. haemolytica and H. somnus in their serum (P < 0.05), colostrum(P < 0.05), and serum of their calves at 3 days and 1 month of age (P < 0.05). There was no significant(P > 0.05) difference in antibody titers in the colostrum and serum of calves from single or double vaccinated dams. Calves vaccinated at 1 and 2 months of age in the face of maternal antibodies toP. haemolytica and H. somnus had significantly(P < 0.05) higher antibodies to P. haemolytica and H. somnus at 4 and 6 months of age than did unvaccinated calves. Calves vaccinated at 3 and 4 months of age in the face of low levels of preexisting antibodies had significantly (P < 0.05) higher antibodies toP. haemolytica at 5 months of age and to H. somnus at 5 and 6 months of age than did unvaccinated calves. Calves vaccinated once at 4 months of age had significantly(P < 0.05) higher antibody titers toP. haemolytica and H. somnus at 4.5 months of age than did unvaccinated calves, but this difference was not apparent at 6 months of age. These results suggest that vaccination of beef cows with a combined Pasteurella haemolytica and Haemophilus somnus vaccine once at 4 weeks prepartum will significantly (P < 0.05) increase passive antibody titers toP. haemolytica and H. somnus in their calves. Double vaccination of calves with preexisting maternal antibodies at 1 and 2 months of age will increase antibody titers to P. haemolytica and H. somnus until 6 months of age. Vaccination of beef calves with low levels of preexisting antibody at 3 and 4 months of age will increase antibody titers to H. somnus until 6 months of age and to P. haemolytica until 5 months of age.However, the level of antibodies achieved by vaccination may depend on the calves being studied, the level of preexisting antibodies, and the efficiency of passive transfer.

Animals↗