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PubMed2002

Genomic scale analysis of Pasteurella multocida gene expression during growth within the natural chicken host.

Little is known about the genomic-scale transcriptional responses of bacteria during natural infections. We used whole-genome microarray analysis to assess the transcriptional state of the gram-negative pathogen Pasteurella multocida, the causative agent of fowl cholera, during infection in the natural chicken host. We compared the expression profiles of bacteria harvested from the blood of septicemic chickens experiencing late-stage fowl cholera with those from bacteria grown in rich medium. Independent analysis of bacterial expression profiles from the infection of three individual chickens indicated that 40 genes were differentially expressed in all three individuals, 126 were differentially expressed in two of the three individuals, and another 372 were differentially expressed in one individual. Real-time reverse transcription-PCR assays were used to confirm the expression ratios for a number of genes. Of the 40 genes differentially expressed in all three individuals, 17 were up-regulated and 23 were down-regulated in the host compared with those grown in rich medium. The majority (10 of 17) of the up-regulated genes were involved in amino acid transport and metabolism and energy production and conversion, clearly indicating how P. multocida alters its biosynthetic and energy production pathways to cope with the host environment. In contrast, the majority (15 of 23) of down-regulated genes were of unknown or poorly characterized functions. There were clear differences in gene expression between the bacteria isolated from each of the three chickens, a finding consistent with individual host variation being an important factor in determining pathogen gene expression. Interestingly, bacteria from only two of the three infected animals had a gene expression profile highly similar to that observed during growth under iron-limiting conditions, suggesting that severe iron starvation may not always occur during P. multocida infection.

Animals↗
PubMed2003

Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens.

Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.

Animals↗
PubMed2004

A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence.

Pasteurella multocida is the causative agent of fowl cholera in birds. In a previous study using signature-tagged mutagenesis, we identified a mutant, AL251, which was attenuated for virulence in mice and in the natural chicken host. Sequence analysis indicated that AL251 had an insertional inactivation of the gene waaQ(PM), encoding a putative heptosyl transferase, required for the addition of heptose to lipopolysaccharide (LPS) (M. Harper, J. D. Boyce, I. W. Wilkie, and B. Adler, Infect. Immun. 71:5440-5446, 2003). In the present study, using mass spectrometry and nuclear magnetic resonance, we have confirmed the identity of the enzyme encoded by waaQ(PM) as a heptosyl transferase III and demonstrated that the predominant LPS glycoforms isolated from this mutant are severely truncated. Complementation experiments demonstrated that providing a functional waaQ(PM) gene in trans can restore both the LPS to its full length and growth in mice to wild-type levels. Furthermore, we have shown that mutant AL251 is unable to cause fowl cholera in chickens and that the attenuation observed is not due to increased serum sensitivity.

Animals↗
PubMed1973

Growth of Pasteurella multocida in vaccinated and normal mice.

Specific pathogen-free CD-1 mice are highly susceptible to infection by Pasteurella multocida strain 5A whether introduced intravenously, intraperitoneally, subcutaneously, or aerogenically. The growth of the challenge organism in the blood, liver, spleen, lung, and peritoneal cavity was quantitated hourly for up to 12 h. Unvaccinated mice died 9 to 12 h after intravenous challenge due to the uncontrolled growth of the organism in all tissues tested. The rate of removal of the bacteria from the blood and of phagocytosis by peritoneal macrophages was extremely slow. In the absence of specific opsonins, more than 90% of the unopsonized challenge inoculum remained in the extracellular growth phase throughout the challenge period. Vaccination of mice with two doses of 10(8) heat-killed (60 C for 60 min) P. multocida given 7 days apart protected the mice against 100 to 1,000 lethal challenge doses. Survival data and growth curves obtained for both actively and passively immunized mice indicated that a humorally mediated immune mechanism was involved. Peak resistance to challenge occurred 21 to 28 days after the mice received the second dose of antigen, and this correlated with an 8- to 16-fold increase in specific agglutinin titers over the same time. Resistance to aerogenic challenge by vaccinated mice was less effective than when other routes of infection were used. The significance of these findings is discussed.

Aerosols↗
PubMed1997

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73.

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.

Amino Acid Sequence↗
PubMed2001

Characterization of a novel transferrin receptor in bovine strains of Pasteurella multocida.

Analysis of bovine respiratory isolates of Pasteurella multocida demonstrated that six of nine strains tested were capable of growth dependent upon bovine transferrin and of specifically binding ruminant transferrins. A single 82-kDa protein was affinity isolated from the P. multocida strains with immobilized bovine transferrin. In contrast to what has been observed in other species, binding of this protein to immobilized transferrin was specifically blocked by the N-lobe subfragment of bovine transferrin. A single gene encoding the 82-kDa protein was flanked by a leucyl-tRNA synthetase gene and an IS1060 element, in contrast to other species where genes encoding the two receptor proteins (TbpB and TbpA) are found in an operonic arrangement. A similar gene arrangement was observed in all of the receptor-positive strains, in spite of the observation that they belonged to different genomic groups. Analysis of the deduced amino acid sequence of the receptor protein indicated that it is a member of the TonB-dependent outer membrane receptor family, and although it is related to transferrin and lactoferrin receptor proteins (TbpAs and LbpAs) from other species, it differs substantially from other members of this group. Amino acid alignments suggest that the reduced size (20 kDa smaller) of the P. multocida TbpA is primarily due to the absence of larger predicted external loops. Collectively these results suggest that P. multocida has a single, novel receptor protein (TbpA) that is capable of efficiently mediating iron acquisition from bovine transferrin without the involvement of a second receptor protein (TbpB).

Amino Acid Sequence↗
PubMed2005

The Pasteurella multocida nrfE gene is upregulated during infection and is essential for nitrite reduction but not for virulence.

Pasteurella multocida is the causative agent of a range of diseases with economic importance in production animals. Many systems have been employed to identify virulence factors of P. multocida, including in vivo expression technology (IVET), signature-tagged mutagenesis, and whole-genome expression profiling. In a previous study in which IVET was used with P. multocida, nrfE was identified as a gene that is preferentially expressed in vivo. In Escherichia coli, nrfE is part of the formate-dependent nitrite reductase system involved in utilizing available nitrite as an electron accepter during growth under anaerobic conditions. In this study, we constructed an isogenic P. multocida strain that was unable to reduce nitrite under either aerobic or anaerobic conditions, thereby demonstrating that P. multocida nrfE is essential for nitrite reduction. However, the nrfE mutant was still virulent in mice. Real-time reverse transcription-PCR analysis indicated that nrfE was regulated independently of nrfABCD by an independent promoter that is likely to be upregulated in vivo.

Animals↗
PubMed1975

Characteristics of Pasteurella multocida of human origin.

Physiological, serological, morphological, and cultural differences were observed among 30 Pasteurella multocida cultures of human origin. The usual variations in the fermentation of glycerol, lactose, sorbitol, trehalose, and xylose were observed. Unlike most P. multocida, two cultures did not produce indol. Six serotypes were found. In addition to the widely recognized iridescent, blue, and watery mucoid (circular) colonies, punctiform colonies were observed. None of the cultures were pathogenic for turkeys. Results of the study indicate that one should be aware of the many variable characteristicx of P. multocida of human origin to facilitate indentification.

Animals↗
PubMed1979

Differentiation of Pasteurella haemolytica biotypes A and T with growth inhibitors.

Differentiation of type A and type T cultures of Pasteurella haemolytica was accomplished by paper disks containing 0.3 microgram of penicillin G, which produced zones of inhibition larger than 10 mm with type A but not with type T strains. Basic fuchsin (0.2 microgram/ml), brilliant green (0.005 microgram/ml), methylene blue (3.1 micrograms/ml) in brain heart infusion broth permitted the growth of type T but that of type A isolates.

Animals↗
PubMed1979

CO2 requirement for growth of a Pasteurella strain isolated from rabbits.

A Pasteurella strain with unusual growth requirements was isolated from a rabbit abscess. Copious growth of this organism was observed only on media supplemented with serum and incubated in the candle jar. Under anaerobic conditions, growth was moderate on enriched media. The organism did not grow on unsupplemented media under these conditions or in an aerobic environment on enriched media.

Abscess↗
PubMed1980

Hyaluronidase production by type B Pasteurella multocida from cases of hemorrhagic septicemia.

Seventy-four cultures of Pasteurella multocida representing all four capsular types, A, B, D, and C, from various animal species and diseases were examined for the production of hyaluronidase by two procedures. In one, hyaluronidase production was determined by the depolymerization of streptococcal capsular hyaluronic acid, and in the other, production was determined by degradation of sodium hyaluronidate in a solid culture medium. Hyaluronidase production was only demonstrated in the 13 type B cultures that had been recovered from cases of hemorrhagic septicemia.

Animals↗
PubMed1981

Cellular fatty acid composition of organisms frequently associated with human infections resulting from dog bites: Pasteurella multocida and groups of EF-4, IIj, M-5, and DF-2.

The cellular fatty acid composition of Pasteurella multocida and four unclassified groups of gram-negative bacteria (EF-4, M-5, IIj, and DF-2) which are frequently associated with human dog-bite infections was determined by gas-liquid chromatography. Strains of P. multocida were readily distinguished from the unclassified groups by the presence of 3-hydroxy myristic acid (3-OH- 14:0). Groups M-5 and EF-4 were characterized by the presence of 3-hydroxy lauric (3-OH-12:0) acid. Only group EF-4 organisms contained 2-OH-16:0, a-17:0, and 17:0 cyclopropane acids. Groups IIj and DF-2 differed from the other groups by the presence of large amounts of a branched-chain 15-carbon acid (i-15:0); they differed from each other by the presence of i-2-OH-15:0 and i-17:1 acids in IIj, which were absent in DF-2. The data indicate that gas-liquid chromatographic analysis for cellular fatty acids provides an additional test for rapid differentiation of these gram-negative organisms associated with dog-bite infections. Similarities observed in the fatty acid compositions of Flavobacterium, IIj, and DF-2 suggest that these two unclassified groups may be additional species of Flavobacterium.

Animals↗
PubMed1983

Capsular and somatic serotypes of Pasteurella multocida isolates recovered from healthy and diseased rabbits in Texas.

A total of 111 Pasteurella multocida isolates recovered from healthy and diseased rabbits were typed for capsular and somatic antigens by the typing systems of Carter and Heddleston, respectively. The major serotypes of the 48 P. multocida isolates recovered from nasal cavities of healthy rabbits were serotypes 12:A (33%), nontypable:A (50%), and nontypable:D (10%). Similarly, the major serotypes of the 63 P. multocida isolates obtained from rabbits with rhinitis, pneumonia, conjunctivitis, tympanitis, or cutaneous abscesses were serotypes 12:A (32%), nontypable:A (30%), and 3:A (16%). Serotype 12:A was predominant, regardless of whether the isolates were recovered from healthy or diseased rabbits.

Animals↗
PubMed1983

Use of a fluorometric immunoassay to determine antibody response to Pasteurella haemolytica in vaccinated and nonvaccinated feedlot cattle.

A retrospective study of the antibody response to Pasteurella haemolytica was conducted by using sera from 368 feedlot cattle divided among five experiments. In three experiments, live vaccines or a bacterin were administered to some of the cattle and others were left as nonvaccinated controls. In two experiments, cattle were not vaccinated. Clinical signs of disease with subsequent recovery developed in 48.0% of the cattle, and 10.3% of the cattle died. Vaccination had no apparent effect on morbidity or mortality. At the time of purchase, 78% of the cattle had low antibody titers (less than 25) as measured by a quantitative fluorometric immunoassay. In most groups of cattle (both vaccinated and nonvaccinated), there was a significant rise in mean antibody titers between the time of purchase and days 28 to 32 in the feedlot. The antibody titers at the time of shipment and health status of cattle. The antibody ratios were significantly greater for cattle that became sick and then recovered compared with those of cattle that remained healthy. Although significance could not be established, antibody titers at the time of shipment were higher for cattle that remained healthy compared with cattle that became sick and then recovered.

Animals↗
PubMed1985

Enteritis caused by Pasteurella pneumotropica infection in hamsters.

Pasteurella pneumotropica was isolated in essentially pure cultures from the bowels of hamsters with enteritis 7 days after parturition. Newly received hamsters showed presence of P. pneumotropica in their nasal cavities but not in their uteri, lungs, spleens, or bowels.

Animals↗
PubMed1986

Enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella haemolytica cytotoxin (leukotoxin) in cattle.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of bovine serum antibodies to the cytotoxin (leukotoxin) of Pasteurella haemolytica. A partially purified, cytotoxic, and immunogenic protein obtained from supernatants of logarithmic-phase P. haemolytica was used as the ELISA antigen. Preadsorption of sera with various cytotoxic, somatic, and capsular antigen preparations demonstrated that the assay was specific for anticytotoxin antibodies. ELISA anticytotoxin titers had a strong, significant correlation to cytotoxin-neutralizing-antibody titers. The ELISA, however, was more rapid and allowed for greater numbers of samples to be run than did the neutralization technique. ELISA anticytotoxin titers were high in cattle vaccinated with a live P. haemolytica vaccine, whereas unvaccinated cattle and cattle receiving a P. haemolytica bacterin had low ELISA anticytotoxin titers. A significant positive correlation between ELISA titers and resistance to experimental bovine pneumonic pasteurellosis was present.

Animals↗
PubMed1987

Hyperimmune serum from rabbits immunized with potassium thiocyanate extract of Pasteurella multocida protects against homologous challenge.

Hyperimmune rabbit sera directed to the KSCN extract of 3:A Pasteurella multocida were characterized by enzyme-linked immunosorbent assay (ELISA), presolubilized cell radioimmunoprecipitation, and immunoblotting analysis. The results showed that the hyperimmune serum had a very high titer of immunoglobulin G ELISA antibody and a negligible immunoglobulin A ELISA antibody, precipitated 10 different outer membrane protein antigens by radioimmunoprecipitation, and reacted to 10 different membrane vesicle antigens of P. multocida by immunoblotting analysis. The hyperimmune rabbit sera were also evaluated for protective efficacy against experimental rabbit pasteurellosis by homologous challenge. Thirty-six rabbits were divided into four groups. Group 1, 2, and 3 rabbits were inoculated intranasally with hyperimmune rabbit serum, phosphate-buffered saline, or normal rabbit serum, respectively, at 24 h prior to and 24, 48, and 72 h after intranasal challenge with the virulent homologous P. multocida strain. Group 4 rabbits were inoculated with normal rabbit serum without challenge. Necropsies of surviving rabbits were performed 2 weeks postinfection. The mortality rates for groups 1 through 4 were 25% (3 of 12), 67% (8 of 12), 75% (6 of 8), and 0% (0 of 4), respectively. The prevalence and severity of pneumonia were significantly lower in the hyperimmune serum-treated rabbits. The prevalence of P. multocida colonization in lungs was significantly lower in group 1 rabbits, and the geometric mean CFU of P. multocida in lungs was 59,166-fold less in group 1 rabbits than in group 3 rabbits. The geometric mean CFU of P. multocida in nasal cavities of group 1 rabbits was significantly lower than that of group 3 rabbits. All challenged rabbits (groups 1,2, and 3) had elevated nasal immunoglobulin A and pulmonary (lung lavage) immunoglobulin A antibody levels at necropsy (day 14 postinfection). Similarly, all challenged rabbits had elevated levels of ELISA immunoglobulin G antibody in serum at day 14 but not at day 7 postinfection, indicating that rabbits receiving hyperimmune serum can mount a specific humoral immune response against the homologous challenge P. multocida organisms. We concluded that hyperimmune serum directed to the KSCN extract of 3:A P. multocida provides significant protection against homologous challenge in rabbits.

Animals↗