Biomedical subjects

S K Maheswaran

Publications and source records attributed to S K Maheswaran.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vitro expression and inhibition of procoagulant activity produced by bovine alveolar macrophages and peripheral blood cells.

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mumol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mumol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.

Animals↗

PubMed1996

Evaluation of three experimental subunit vaccines against pneumonic pasteurellosis in cattle.

The objective of this study was to evaluate the efficacy of three Pasteurella haemolytica A1 derived experimental subunit vaccines against pneumonic pasteurellosis in cattle. The three vaccines were: (a) culture supernatant (CS) containing leukotoxin (Lkt), lipopolysaccharide (LPS) and capsular polysaccharide (CP); (b) sodium salicylate extract (SSE) containing iron regulated outer membrane proteins (IROMPs), LPS and CP; (c) and a combination of the above two. Vaccine efficacy was defined in terms of reduction in clinical and pneumonic lesion scores after intrapulmonic challenge with live P. haemolytica. The results indicate that the CS vaccine elicited antibodies against both Lkt and CP, while the SSE vaccine elicited antibodies against IROMPs and CP. Animals inoculated with the combination vaccine showed increased levels of antibodies against IROMPs, Lkt and CP. There was significant correlation between lung and serum antibodies against Lkt, CP and IROMPs. Animals that received the combination vaccine had significantly lower mean pneumonic lung score as compared to SSE and control groups. The animals which received CS vaccine had mean pneumonic lung score significantly lower than that of control group. A strong negative correlation existed between serum antibody levels against Lkt, IROMPs, CP and pneumonic lung scores. The results from this study demonstrate the usefulness of CS vaccine alone or in combination with SSE vaccine in bringing about optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis.

Animals↗

PubMed1996

Comparative evaluation of antibodies induced by commercial Pasteurella haemolytica vaccines using solid phase immunoassays.

The objective of this study was to evaluate the ability of four commercial vaccines to elicit antibodies against the leukotoxin (Lkt), capsular polysaccharide (CP), iron regulated outer membrane proteins (IROMPs), and whole cell (WC) antigens of Pasteurella haemolytica A1. Modified double antibody sandwich enzyme linked immunosorbent assays (ELISAs) were developed to measure antibody levels against Lkt, CP and IROMPs. An indirect ELISA was developed to measure the levels of antibody against WC antigens. The ideal cut off points for ELISAs were determined on receiver operating characteristic curves, using sera from 30 calves injected subcutaneously with a live P. haemolytica 12296 strain as positive control and sera from 30 colostrum-deprived calves as negative control. The vaccines evaluated were: 'One Shot' (SmithKline Beecham, West Chester, PA) a bacterin-toxoid, 'Presponse' (Langford Laboratories, Guelph, Ontario) a Lkt-rich culture supermatant, 'Once PMH' (BioCor Inc., Omaha, NE) a modified live vaccine, and 'Septimune' (Fort Dodge laboratories, Fort Dodge, IA) an outer membrane extract. Thirty, 4-6 week old Holstein calves were randomized into 5 groups to receive one of the four vaccines or a placebo (sterile phosphate buffered saline). The calves were vaccinated intramuscularly on day 0 and on day 14, and bled on days, 0, 14, and 28 to measure antibody levels against Lkt, CP, IROMPs, and WC antigens of P. haemolytica Al. 'One Shot', and 'Once PMH' vaccinates showed a significant (P < 0.05) increase in antibody levels against Lkt at 28 days. 'Once PMH' vaccinates also showed significant (P < 0.05) increase in antibody levels against IROMPs at 28 days compared to the other four groups but this increase was not significant over time within the 'Once PMH' group. 'Presponse', 'Once PMH' and 'One Shot' vaccinates showed a significant (P < 0.05) increase in antibody levels against CP over time. These groups also had significantly higher antibody levels against CP, compared to controls and 'Septimune' vaccinates at 14 and 28 days (P < 0.05).

Animals↗

PubMed1996

Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis.

The objective of this study was to evaluate the efficacy of three commercial vaccines against experimental pneumonic pasteurellosis in cattle. The three vaccines were: (a) One Shot (SmithKline Beecham, West Chester, PA.), (b) Presponse (Langford Laboratories, Guelph, Ontario) and (c) Once PMH (BioCor, Omaha, NE.). Protective immunity was evaluated in terms of lower clinical and pneumonic lesion scores after endobronchial challenge with virulent P. haemolytica. The results indicate that One Shot elicited antibodies against leukotoxin (Lkt), capsular poly-saccharide (CP) and surface antigens (SA), while Presponse and Once PMH elicited antibodies against CP and SA. There was significant correlation between lung and serum antibody levels against Lkt (P < 0.0001), CP (P < or = 0.0001) and IROMPs (P < or = 0.035). Animals that received the One Shot had significantly (P < or = 0.05) lower mean pneumonic lesion score (36.6 +/- 10.97) as compared to the control group (48.6 +/- 25.92). A significant negative correlation (-0.41; P < or = 0.008) existed between serum antibody levels against Lkt and pneumonic lesion score. High serum antibodies against SA did not correlate with reduction in pneumonic lesion score. In addition, high antibody levels against CP did not correlate consistently with reduced pneumonic lesion scores. The results from this study demonstrates that commercial vaccines evaluated in this trial did not confer optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis. However, One Shot vaccinates showed a better protective immunity compared to the other two vaccine groups.

Animals↗

PubMed1995

Increased tumor necrosis factor-alpha and interleukin-1 beta expression in the lungs of calves with experimental pneumonic pasteurellosis.

We used a well characterized pneumonic pasteurellosis model in calves to determine whether increased proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) expression and secretion were associated with pneumonic lesions. Bronchoalveolar lavage fluids, lavage cells consisting of alveolar macrophages and neutrophils with degenerative changes, and lung tissues were analyzed for the presence of TNF-alpha and IL-1 beta approximately 48 h following endobronchial inoculation of logarithmic phase Pasteurella haemolytica 12296 organisms. Levels of TNF-alpha and IL-1 beta mRNA were significantly increased in lavage cells of P. haemolytica-infected animals but not in cells from phosphate buffered saline (PBS) inoculated controls based on in situ hybridization analysis. Significantly increased levels of TNF-alpha, and IL-1 beta mRNA were also expressed within the pneumonic lesions from P. haemolytica-infected calves. In contrast, lung tissues from PBS-inoculated control calves had cytokine mRNAs expressed at extremely low levels. Increased levels of bioactive IL-1 and immunoreactive (not bioactive) TNF-alpha were found in lavage fluids from P. haemolytica-infected calves compared with lavage fluids from PBS-inoculated calves. These findings indicate that the proinflammatory cytokines TNF-alpha and IL-1, may be associated with pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Animals↗

PubMed1995

Colorimetric assay using XTT for assessing virulence of avian Pasteurella multocida strains.

A colorimetric assay using sodium 3,3'-[1[(phenylamino)carbonyl]3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) was adapted to quantitate bactericidal activity of chicken macrophage HD 11 cell line against five Pasteurella multocida strains and an avirulent transposon insertion mutant. The strains used were virulent P1059, and D92, and four avirulent strains including a streptomycin resistant mutant of P1059 (P1059 SmR), two live vaccine strains namely, the Clemson University (CU) and M9, and a transposon insertion mutant PmTn-294. Percentage of bacteria killed by chicken macrophage (HD 11) cells was determined by extrapolation from a standard formazan curve derived by incubating XTT with known bacterial cell numbers of each strain. The amount of formazan as measured by absorption at 450 nm was directly related to the number of viable bacterial cells. The percentages of P1059 SmR, CU, M9 and PmTn-294 killed by HD 11 cells were approximately 50%, 61%, 25% and 34%, respectively. By contrast, the virulent P1059 and D92 strains were resistant to killing, and were able to replicate inside the HD 11 cells. Association of virulence with resistance to phagocytic killing by HD 11 cells as assessed by the colorimetric bactericidal assay, was validated with resistance to complement (C')-mediated killing and a turkey mortality test. Strains P1059 and D92 were resistant to C'-mediated killing, whereas strains P1059 SmR, CU, M9 and PmTn-294 strains were susceptible. All turkeys challenged with P1059 or D92 were dead within 18 hrs. Mortality did not occur in turkeys challenged with strains of P1059 SmR, M9 and PmTn-294. The mortality among CU challenged turkeys ranged from 0 to 40%. The results suggest that the colorimetric bactericidal assay using XTT can be used to quantitate chicken macrophage phagocytic killing of P. multocida strains, and may be a valuable assay to differentiate virulent from avirulent strains of avian P. multocida.

Animals↗

PubMed1995

Purified Pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages.

We obtained biologically active purified leukotoxin (Lkt) from Pasteurella haemolytica serotypel, strain 12296 using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three species of Lkt of molecular masses 95, 100, and 104 kDa were obtained. Purity of all three species of Lkt was confirmed by analytical SDS-PAGE and Western blot (immunoblot) analysis. Results from the chromogenic Limulus amebocyte lysate assay and silver staining of SDS-PAGE patterns indicated that the preparations were free of contaminating lipopolysaccharide. We then studied the kinetics of TNF alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNF alpha and IL-1 beta gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNF alpha and IL-1 beta mRNA expression were detectable at 1 h after stimulation with 1 leukotoxin unit/ml. The expression peaked at 2 h, steadily declining up to 6 h, and was undetectable by 10 h. Secreted TNF alpha measured by bioassay peaked at 4-6 h and accumulated at a lesser concentration after 6 h. By contrast, secreted IL-1 peaked at 6 h and decreased significantly by 10 h. The ability of purified Lkt to induce TNF alpha and IL-1 beta gene expression and secretion of bioactive proteins was suppressed by Ca2+ chelating agents, 5 mM EDTA and 5 mM EGTA, but not polymyxin B. Heat-inactivation of the purified Lkt that had lost its cytocidal property completely abrogated induction of TNF alpha and IL-1 beta gene expression and secretion in bovine AMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Induction of inflammatory cytokines in bovine alveolar macrophages following stimulation with Pasteurella haemolytica lipopolysaccharide.

Bovine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) cDNAs were generated by reverse transcription and then by PCR amplification from lipopolysaccharide (LPS)-stimulated alveolar macrophage RNA. The amplified cDNAs were cloned into pPow and expressed in Escherichia coli DH5 alpha. The expressed proteins were confirmed as TNF-alpha and IL-1 beta by Western blot (immunoblot) analysis and bioassays. We then used the cloned genes as probes in Northern (RNA) blots and investigated the kinetics of TNF-alpha and IL-1 beta mRNA expression in bovine alveolar macrophages stimulated with purified LPS from Pasteurella haemolytica 12296. The effect of LPS on TNF-alpha and IL-1 beta gene expression was dose dependent, and induction was observed at a concentration of 0.01 microgram/ml. Both TNF-alpha and IL-1 beta mRNA expression were detectable within 0.5 h after stimulation with 1 microgram of LPS per ml, peaked at 1 to 2 h, steadily declined up to 16 h, and were undetectable by 24 h. Secreted TNF-alpha measured by bioassay peaked at 4 h and accumulated at a lesser concentration in conditioned medium throughout the 24 h. By contrast, secreted IL-1 beta was induced at 8 h and reached a maximal concentration at 24 h after stimulation. The ability of LPS to induce TNF-alpha and IL-1 beta gene expression and secretion of bioactive proteins were suppressed by polymyxin B. Our findings support a role for LPS from P. haemolytica in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.

Animals↗

PubMed1995

Pasteurella haemolytica serotype 2 contains the gene for a noncapsular serotype 1-specific antigen.

An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5 alpha to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1- or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1- and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium.

Amino Acid Sequence↗

PubMed1995

Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae.

An Actinobacillus pleuropneumoniae infection model in swine was established to study the expression of inflammatory cytokines during acute respiratory disease. Lavage fluid, lavage cells consisting primarily of alveolar macrophages, and lung tissue were analyzed for the presence of various cytokines at 2, 4, 8, and 24 h following endotracheal inoculation of A. pleuropneumoniae. Interleukin-1 beta (IL-1) and IL-8 mRNA levels were elevated within 2 h in lavage cells of animals inoculated with A. pleuropneumonia but not in cells from controls treated with saline-bovine serum albumin, based on Northern (RNA blot) analysis. Tumor necrosis factor (TNF) mRNA was present at low levels in all animals, and the level was not increased at any time point. In situ hybridization was more sensitive than Northern blotting and revealed elevations of all three cytokines in lavage cells within 2 to 4 h of A. pleuropneumoniae inoculation. IL-6 was detected in lavage cells by in situ hybridization but not by Northern blotting. In lung tissue obtained 18 to 24 h after A. pleuropneumoniae instillation, all cytokine mRNAs, including that of IL-6, were detected by Northern blot analysis. The levels of bioactive IL-1 and IL-6 in lavage fluids increased approximately 1,000-fold following A. pleuropneumoniae inoculation, but TNF bioactivity was not detected. Morphological localization of cytokine mRNAs by in situ hybridization indicated markedly increased levels of TNF, IL-1, and IL-8 mRNAs at the periphery of focal lung lesions. These findings indicate that inflammatory cytokines, particularly IL-1 and IL-8, are associated with the development of pleuropneumonia and may contribute to disease severity.

Actinobacillus Infections↗

PubMed1995

Expression of iron-regulated outer membrane proteins by porcine strains of Pasteurella multocida.

The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.

Animals↗

PubMed1993

Enhancement of neutrophil-mediated injury to bovine pulmonary endothelial cells by Pasteurella haemolytica leukotoxin.

In this study, we used an in vitro coculture system to determine which virulence factor from Pasteurella haemolytica A1 was responsible for augmenting bovine polymorphonuclear neutrophil (PMN)-mediated killing of bovine pulmonary artery endothelial cells (BPAEC). A 51Cr release cytotoxicity assay was used as a measure of BPAEC killing. The mechanisms associated with this BPAEC killing were also studied. Our results demonstrated that the leukotoxin and not the lipopolysaccharide from P. haemolytica was responsible for augmenting the PMN-mediated killing of BPAEC. Furthermore, this augmented killing was related to the stimulation of PMNs by the leukotoxin. Killing of BPAEC by leukotoxin-stimulated PMNs was diminished in the presence of the H2O2 inactivator, catalase. The membrane-permeant H2O2, hydroxyl radical (HO.) scavenger 1,3-dimethyl-2 thiourea, and the HO. scavenger dimethyl sulfoxide but not the myeloperoxidase inhibitor sodium azide attenuated this BPAEC killing. Pretreatment of BPAEC with a 21-aminosteroid (U74500A), a potent iron chelator-antioxidant, provided the most effective protection against BPAEC killing induced by leukotoxin-stimulated PMNs. These data were compatible with the concept that the H2O2 generated by leukotoxin-stimulated PMNs interacts with intracellular iron in the endothelial cell to form highly reactive HO.. We suggest that HO. may be a key factor in BPAEC killing. Furthermore, since the elastase-specific inhibitor N-methoxy-succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) also attenuated BPAEC killing and both CMK and 1,3-dimethyl-2 thiourea functioned additively in protecting against BPAEC killing, we conclude that both HO. and elastase may jointly contribute to BPAEC killing induced by leukotoxin-stimulated PMNs. This study broadens our understanding of how leukotoxin-stimulated PMNs injure lung endothelial cells and provides new insight into the pathogenesis of bovine pneumonic pasteurellosis.

Amino Acid Sequence↗

PubMed1992

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.

Animals↗

PubMed1991

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Animals↗

PubMed1991

Morphological and morphometrical analysis of the acute response of the bovine alveolar wall to Pasteurella haemolytica A1-derived endotoxin and leucotoxin.

Endotoxin or leucotoxin derived from Pasteurella haemolytica biotype A serotype 1 or saline was deposited by fibreoptic bronchoscopy into the caudal segment of the right anterior lung lobe of calves, and the lesions were characterized by light and transmission electron microscopy. Morphometric techniques were used to determine if changes in the arithmetic mean thickness of the alveolar wall occurred. Group 1 calves (n = 2) were inoculated with 6 ml saline, groups 2 calves (n = 3) received 6 ml of a partially purified leucotoxin preparation, group 3 calves (n = 3) received 96 micrograms of endotoxin in 6 ml of saline and group 4 calves (n = 3) received 2.5 mg of endotoxin in 6 ml of saline. Calves were killed 4 h after inoculation. Lesions in groups 2, 3 and 4 were similar and we found that (a) endotoxin alone is capable of initiating an inflammatory response in the bovine lung, (b) leucotoxin causes cytotoxic changes in alveolar macrophages but not in parenchymal cells of the lung, (c) neutrophil sequestration and platelet aggregation occur in alveolar capillaries in association with pulmonary intravascular macrophages, (d) neutrophils and fibrin were found in the alveolus in close association with alveolar macrophages, (e) disruption of the alveolar epithelial layer occurred in association with neutrophils and (f) there were no significant increases in the arithmetic mean thickness of the alveolar wall.

Animals↗

PubMed1991

Genomic distribution of a serotype 1-specific antigen-coding DNA fragment of Pasteurella haemolytica.

A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.

Animals↗

PubMed1991

Alterations in pulmonary morphology and peripheral coagulation profiles caused by intratracheal inoculation of live and ultraviolet light-killed Pasteurella haemolytica A1 in calves.

Eighteen male Holstein calves were divided into groups of three and inoculated intratracheally with 5 x 10(9) logarithmic phase or ultraviolet light-killed Pasteurella haemolytica biotype A serotype 1. Serial coagulation profiles were done on one calf from each group during the first 24 hours after inoculation. One calf from each group was necropsied at 4, 12, and 24 hours after inoculation and lesions were characterized with light and transmission electron microscopy. We found that 1) the pulmonary intravascular macrophage may have an important role in the early intravascular inflammatory events; 2) there was morphologic evidence for local initiation of the coagulation cascade in the lung early in the disease process but it was not a consumptive process; and 3) killed-bacteria were capable of causing fibrin exudation, platelet aggregation and alveolar epithelial damage similar to live bacteria, but the degenerative changes in neutrophils, endothelial cells and intravascular fibrin formation that occur with live bacteria were not seen.

Animals↗

PubMed1991

Changes in blood and bronchoalveolar lavage fluid components in calves with experimentally induced pneumonic pasteurellosis.

Pneumonic pasteurellosis was experimentally induced in calves by inoculation of 5 x 10(8) Pasteurella haemolytica organisms into the right diaphragmatic lung lobe. Blood and bronchoalveolar lavage fluid samples were obtained prior to inoculation and at postinoculation hour (PIH) 2, 4, and 6. Calves developed acute lung injury, characteristic of pneumonic pasteurellosis. Lesions were found only in the right diaphragmatic lobe. By PIH 4, significant (P less than 0.01) increases were detected in lavage fluid total cell count, neutrophil count, total protein and albumin concentrations, and alkaline phosphatase (ALP) and lactic dehydrogenase (LD) activities. Myeloperoxidase and elastase activities did not increase. Neutrophil depletion ameliorated the lung lesions and prevented the increase in lavage fluid cell count, total protein, and albumin concentrations and ALP and LD activities. Treatment with the iron chelator, deferoxamine mesylatehydroxyethyl starch, attenuated the increase in total protein and albumin concentrations and ALP and LD activities at PID 4, but not PIH 6. Treatment with a neutrophil function inhibitor, pentoxifylline, prevented the increase in lavage fluid neutrophil numbers, but accentuated the increase in total protein and albumin concentrations, and ALP, LD, myeloperoxidase, and elastase activities.

Animals↗