Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.     

Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera

A 5' Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5' Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5' Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.     

Constitutive expression of Pasteurella multocida toxin

Abstract The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of β-galactosidase from a cps-lacZ fusion ( lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.     

Cloning and expression of the dermonecrotic toxin gene of Pasteurella multocida ssp. multocida in Echerichia coli

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.     

A minimal medium for growth of Pasteurella multocida

Abstract We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm ). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.     

Fowl cholera outbreak in domestic poultry and epidemiological properties of Pasteurella multocida isolate

Symptoms of fowl cholera including orofacial edema, swollen and edematous wattles and combs, and severe respiratory disorders were detected in domestic poultry in two broiler breeder farms: one located in Gyeong-gi Province (October, 2000) and the other in Chung-cheong-nam Province (March, 2001). Gram-negative, bipolar staining bacillus was easily found in a direct smear. The biochemical properties of isolates were examined using a standard diagnosis method, proving that they were 99.7% similar to the Pasteurella multocida (P. multocida: PM), a pathogenic and causative agent of fowl cholera (FC). As a result, an FC outbreak in domestic fowls was confirmed for the first time in Korea since 1942. Because FC was detected in broiler breeder farms for the first time in 59 years at the same time as an FC outbreak was confirmed in wild birds (October, 2000), our concern was focused on whether the PM strains that originated in wild birds were transmitted into poultry farms. The possibility was tracked down by comparing phenotypic and genetic properties between the two types of PM strains. PM strains of chicken origin showed prominent differences from the PM strains of wild bird origin in both phenotypic and genetic properties. An examination of the origin of the wild bird bacteria was conducted, but no evidence has been identified that PM strains from the wild bird were introduced into domestic poultry farms.     

Serological types of Pasteurella multocida isolated from turkeys and chickens in Canada

Outbreaks of fowl cholera continue to plague the Canadian poultry industry despite widespread immunization against the causative agent, Pasteurella multocida. Fowl cholera bacterins currently employed by domestic poultry growers contain three serological types, namely, serotypes 1, 3, and 4. In this study a total of 84 strains of P. multocida were isolated in Canada from outbreaks of fowl cholera in turkeys and chickens. Serotyping was accomplished using the gel diffusion precipitin test. Based on the gel diffusion precipitation patterns, 27 serotypes containing one to six antigenic determinants were recognized. The most prevalent serotype both in turkeys and chickens appeared to be type 3. Significantly, greater than 20% of P. multocida isolates failed to react with antisera raised against serotypes 1, 3, and 4.     

Pasteurella multocida from outbreaks of avian cholera in wild and captive birds in Denmark

An outbreak of avian cholera was observed among wild birds in a few localities in Denmark in 2001. The highest mortalities were among breeding eiders (Somateria mollissima) and gulls (Larus spp.). Pulsed-field gel electrophoresis (PFGE) was conducted using ApaI and SmaI as restriction enzymes and restriction enzyme analysis (REA) using HpaII. The Pasteurella multocida subsp. multocida strain isolated from birds in this outbreak was indistinguishable from a strain that caused outbreaks in 1996 and 2003. Most isolates from domestic poultry had other PFGE patterns but some were indistinguishable from the outbreak strain. Among 68 isolates from wild birds, only one PFGE and one REA pattern were demonstrated, whereas among 23 isolates from domestic poultry, 14 different SmaI, 12 different ApaI, and 10 different HpaII patterns were found. The results suggest that a P. multocida strain has survived during several years among wild birds in Denmark.     

Interaction of Pasteurella multocida with free-living amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Interaction of Pasteurella multocida with Free-Living Amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Importance of the galE gene on the virulence of Pasteurella multocida

The galE gene of Pasteurella multocida has been isolated by complementing galE-defective mutants of Salmonella typhimurium with a plasmid library of this organism. The complete nucleotide sequence of the P. multocida galE gene consists of 1017 nucleotides, encoding a predicted polypeptide of 339 amino acids. The deduced amino acid sequence displayed the highest identity (85%) to the GalE protein of Haemophilus influenzae. However, the gene organization surrounding the galE locus was different from that of H. influenzae. A galE-defective mutant of P. multocida was obtained by replacement of the active galE gene by a copy inactivated in vitro. The resulting galE mutant was highly attenuated as seen in a biological test carried out in a mouse model.     

Immunization of Mice with Components of Pasteurella multocida

Log-phase cells of Pasteurella multocida strain P-1059 were used to prepare isolated culture filtrate, cell wall, and cytoplasmic components. Culture filtrate was further separated by column chromatography. A portion of cytoplasm and culture filtrate was conjugated to ferritin by means of metaxylylene diisocyanate. Cell walls induced more protection in mice than the conjugated or unconjugated cytoplasm or culture filtrate. The cell walls caused edema and erythema when given intradermally in rabbits, whereas cytoplasm and culture filtrate produced dermal necrosis. The first of four chromatographically separated fractions of culture filtrate was possibly more immunogenic in mice than cell walls. This fraction was less reactive intradermally in rabbits than cell walls but more reactive than the other fractions.     

Kinetics of expression of connective tissue growth factor gene during liver regeneration after partial hepatectomy and D-galactosamine-induced liver injury in rats

Connective tissue growth factor (CTGF) is up-regulated by TGF-beta1 during wound healing. The present study examined the expression of CTGF during regeneration after 70% partial hepatectomy (PH) or d-galactosamine (GalN)-injured liver in rats. CTGF, TGF-beta1, and type I collagen mRNAs were semiquantified by a ribonuclease protection assay. After PH, TGF-beta1 and type I collagen were increased at 2-6 h and at 12-48 h. CTGF increased at 6 h and returned to the control level thereafter. The ribonuclease protection assay of cultured hepatic stellate cells (HSC) and in situ hybridization suggest that the cells express CTGF along sinusoid might be HSCs. After GalN administration, CTGF increased at 2-96 h with a shoulder peak at 6-12 h followed by a main peak at 24 h. TGF-beta1 and type I collagen were up-regulated with kinetics similar to those of CTGF. The different kinetics between PH and GalN regenerations indicate that regulation of CTGF in the two processes is different. Higher TGF-beta1 expression after inflammatory/necrotic process in the GalN regeneration may caused the prolonged CTGF expression.     

Effects of antibiotics on the growth and morphology of Pasteurella multocida.

The effects of subminimal inhibitory concentrations (subMICs) of certain antibiotics, namely penicillin G, tetracycline and trimethoprim/sulphamethoxazole, on the growth and morphology of Pasteurella multocida were evaluated. SubMICs of penicillin markedly reduced the growth of P. multocida. Tetracycline and trimethoprim/sulphamethoxazole had no effect on its growth. SubMICs of penicillin greatly affected the morphology of P. multocida. At the highest concentrations tested (1/2 and 1/4 MIC) cells were acapsulate, and long filamentous cells (4-6 microns) were observed with some isolates. There was no correlation between the observed differences in the penicillin-binding proteins of the P. multocida isolates, and the extent of cell filamentation induced by penicillin G. SubMICs of tetracycline and trimethoprim/sulphamethoxazole did not seem to affect capsule production although filamentation was observed. Our results indicate that subMICs of penicillin can reduce growth of P. multocida. Furthermore, results also indicate that subMICs of antibiotics can affect the production of capsular material and the morphology of P. multocida.     

Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant

The aroA gene from Pasteurella multocida was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The nucleotide sequence of the P. multocida aroA gene indicated an open reading frame encoding a protein of 441 amino acids, which showed a high degree of homology with the amino acid sequences of various other bacterial AroA proteins. The cloned P. multocida aroA gene was inactivated by insertion of a kanamycin-resistance gene and reintroduced by allelic exchange into the chromosome of P. multocida using the suicide vector pJM703.1. The P. multocida aroA mutant was highly attenuated in a mouse model. Mice immunized intraperitoneally with two doses of live P. multocida aroA mutant were completely protected against a lethal parental strain challenge.