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Biomedical subjects

A A Potter

Publications and source records attributed to A A Potter.

At least 19 recordsLinked to original sources

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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