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.     

Specific Detection of Pasteurella multocida in Chickens with Fowl Cholera and in Pig Lung Tissues Using Fluorescent rRNA In Situ Hybridization

A Pasteurella multocida species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida.     

Physiological and serological characteristics of 48 Pasteurella multocida cultures from rabbits

Forty-eight Pasteurella multocida cultures collected from rabbits over a 56-year period were examined to determine their physiological characteristics and to determine their serological types in the gel diffusion precipitin test. Generally, the physiological characteristics from 30 tests were typical for P. multocida. There were a few atypical variations in the fermentation of lactose and maltose and variations in trehalose, dulcitol, xylose, sorbitol, and glycerol. Seven cultures did not produce indole, and four cultures did not produce detectable amounts of hydrogaen sulfide. In obliquely transmitted light, 26 cultures formed large, slightly iridescent, mucoid colonies, 17 cultures had iridescent colonies, and 5 cultures had blue colonies. Heat-stable antigens from the 48 cultures reacted with antisera prepared from P. multocida type cultures presenting serotypes 1, 3, 4, 12, and 15. Antigens from 15 cultures reacted slightly with antisera from more than one serotype. Overall, gel precipitin reactions involving serotype 3 (25%) and serotype 12 (66.7%) were the most prevalent.     

Evidence for non-siderophore-mediated acquisition of transferrin-bound iron by Pasteurella multocida.

Two clinical isolates of Pasteurella multocida associated with bovine pneumonia were examined for iron acquisition. Both isolates were capable of obtaining iron for growth from bovine but not from human, avian, equine or porcine transferrin. This correlated with specific binding of bovine transferrin by iron-limited cells or isolated membranes. No siderophore was detected in the strains by a general screening assay. In response to iron-limited conditions, a number of high molecular mass iron-regulated outer membrane proteins were produced including an 82 kDa receptor protein which was affinity isolated with biotinylated transferrin. In contrast, avian strains of P. multocida could not use transferrin-bound for growth and did not express either transferrin binding activity or the 82 kDa receptor protein.     

Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida.

Two major outer membrane proteins of Pasteurella multocida, designated OmpH and OmpA, were characterized and shown to be related to the families of porin and heat-modifiable proteins, respectively. The backpack hybridoma tumor system in BALB/c mice was used to continuously deliver immunoglobulin G2b (IgG2b) monoclonal antibodies (MAbs) specific for OmpH (MAb MT1) and OmpA (MAb MT4.1). MAbs were detected in serum and peritoneal lavage samples of mice bearing hybridoma tumors by an enzyme-linked immunosorbent assay and an immunoblot assay. Highly significant protection was observed in mice bearing MT1 hybridoma tumors against both intraperitoneal and intranasal challenge infections with homologous nontoxigenic P. multocida strains possessing MAb MT1-reacting epitopes, whereas the mice bearing MT4.1 hybridoma tumors were not protected. The numbers of P. multocida organisms in the lungs of mice bearing MT1 hybridoma tumors were significantly less than those in lungs of mice bearing MT4.1 hybridoma tumors at 48 h postchallenge. These results indicate that the OmpH-specific MAb inhibited proliferation of P. multocida in the lungs. MAb MT1 was unable to kill P. multocida in vitro in the presence of complement. However, an enhanced phagocytosis by polymorphonuclear cells (PMNs) was observed in mice bearing MT1 hybridoma tumors. P. multocida induced a more extensive and rapid influx of PMNs into the peritoneal cavity of mice bearing MT1 hybridoma tumors than of mice bearing MT4.1 hybridoma tumors. The results of this study demonstrate for the first time that IgG MAbs against OmpH of P. multocida are involved in the protection of mice against lethal challenge infection by means of opsonization and inhibition of proliferation of P. multocida as a result of increased influx of PMNs into the infection site.     

A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis.

Monoclonal antibodies (MAbs) directed against the 37.5-kDa outer membrane protein were produced by fusing myeloma cells with spleen cells obtained from mice immunized with a pathogenic strain of Pasteurella multocida isolated from a rabbit. Desirable MAbs were selected by enzyme-linked immunosorbent assay, whole-cell radioimmunoprecipitation (WC-RIP), and Western blot (immunoblot) analysis. WC-RIP and Western blot analyses, using MAb 1608 adsorbed with intact P. multocida cells and the eluted MAb, demonstrated that the antigen recognized by this MAb is exposed on the cell surface, is antibody accessible, and has an estimated molecular mass of 37.5 kDa. Treatment of outer membrane vesicles of P. multocida with proteinase K totally abrogated the MAb 1608 activity, indicating that this MAb binds to a protein antigenic determinant. Furthermore, MAb 1608 was nonreactive to purified lipopolysaccharide in Western blot analysis. Passive transfer studies showed that nine rabbits inoculated intranasally with MAb 1608 and homologously challenged intranasally had significantly reduced mortality, severity of pneumonia, prevalence of P. multocida colonization in nonrespiratory organs, and numbers of P. multocida in nasal cavities compared with the controls. Furthermore, the number of P. multocida in lungs was reduced 84,750-fold. Similarly, passive transfer experiments indicated that MAb 1608 protected mice against homologous and heterologous challenges with P. multocida strains bearing the antigenic determinant recognized by MAb 1608. However, no protection was afforded by MAb 1608 when mice were challenged with a P. multocida strain lacking the antigenic determinant recognized by MAb 1608. This study establishes that the 37.5-kDa outer membrane protein is the target for a protective MAb.     

Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR.

A PCR assay was developed for the differentiation of toxigenic Pasteurella multocida subsp. multocida strains, the major etiologic agent for progressive atrophic rhinitis in pigs, from nontoxigenic strains. The PCR targeted a toxA gene encoding a 143-kDa dermonecrotic toxin that is considered to be the central etiologic factor in progressive atrophic rhinitis. toxA fragments were amplified from toxigenic P. multocida isolates but not from nontoxigenic isolates or other bacteria isolated from pigs. The sensitivity of the reaction was as low as 10 pg of chromosomal DNA from a toxigenic strain. The results obtained by PCR of the DNAs of 187 field isolates of P. multocida were consistent with those obtained by the guinea pig skin test and Western blot (immunoblot) analysis. Restriction fragment analysis of the PCR-amplified fragments from 67 field isolates and comparison of the DNA sequences of fragments from capsular serotype A and D strains suggest that the PCR-amplified region, which is considered to encode the major immunologic determinants of the toxin, would be the same among P. multocida strains. The PCR that we describe should be useful for the diagnosis and the etiologic survey of progressive atrophic rhinitis.     

Novel one-step method for detection and isolation of active-toxin-producing Clostridium difficile strains directly from stool samples

The alarming emergence of hypervirulent strains of Clostridium difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. The C. difficile toxins A and B are critical virulence factors, and strains can either be toxin-producing (virulent) or non-toxin-producing (nonvirulent). Strains that are isolated from human infections generally produce either toxin A or toxin B or both. The methods currently available for culturing C. difficile do not differentiate strains that produce active toxins from strains that do not produce toxins or produce inactive toxins. As a result, the identification and isolation of toxin-producing strains from stool is currently a two-step process. First, the stool is plated on a selective medium, and then suspected colonies are analyzed for toxin production or the presence of the toxin genes. We describe here a novel selective and differential culture method, the Cdifftox plate assay, which combines in a single step the specific isolation of C. difficile strains and the detection of active toxin. This assay was developed based on our recent finding that the A and B toxins of C. difficile cleave chromogenic substrates that have stereochemical characteristics similar to their natural substrate, UDP-glucose. The Cdifftox plate assay is shown here to be extremely accurate (99.8% effective) in detecting toxin-producing strains through the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. The Cdifftox plate assay advances and improves the culture approach such that only C. difficile strains will grow on this agar, and virulent strains producing active toxins can be differentiated from nonvirulent strains, which do not produce active toxins. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains.     

Serological response to Pasteurella multocida NanH sialidase in persistently colonized rabbits

Pasteurella multocida is a mucosal pathogen that colonizes the upper respiratory system of rabbits. Respiratory infections can result, but the bacteria can also invade the circulatory system, producing abscesses or septicemia. P. multocida produces extracellular sialidase activity, which is believed to augment colonization of the respiratory tract and the production of lesions in an active infection. Previously, it was demonstrated that some isolates of P. multocida contain two unique sialidase genes, nanH and nanB, that encode enzymes with different substrate specificities (S. Mizan, A. D. Henk, A. Stallings, M. Meier, J. J. Maurer, and M. D. Lee, J. Bacteriol. 182:6874-6883, 2000). We developed a recombinant antigen enzyme-linked immunosorbent assay (ELISA) based on the NanH sialidase of P. multocida and demonstrated that rabbits that were experimentally colonized with P. multocida produce detectable anti-NanH immunoglobulin M (IgM) and IgG in serum, although they demonstrated no clinical signs of pasteurellosis. In addition, clinically ill pet rabbits infected with P. multocida possessed IgM and/or IgG antibody against NanH. The NanH ELISA may be useful for the diagnosis of P. multocida infections in sick rabbits as well as for screening for carriers in research rabbit colonies.     

Value of Enterobacterial Repetitive Intergenic Consensus PCR for Study of Pasteurella multocida Strains Isolated from Mouths of Dogs

Fifty-six Pasteurella multocida strains (40 P. multocida subsp. septica and 16 P. multocida subsp. multocida strains) isolated from the mouths of 56 dogs among the 134 living in a French canine military training center (132e Groupe Cynophile de l'Armée de Terre, Suippes, France) were studied by use of enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and restriction fragment length polymorphism (RFLP) techniques. Both techniques showed genomic heterogeneity of the strains studied. However, RFLP was more discriminatory than ERIC-PCR for differentiating P. multocida strains. All but three pairs of strains were discriminated by RFLP, suggesting a limited circulation of strains between these dogs living in proximity. Although ERIC-PCR is easier and faster to perform, it cannot be recommended for epidemiological studies of P. multocida strains.     

A group of indole-negative bovine strains with high deoxyribonucleic acid homology to Pasteurella multocida

A group of nine bovine Pasteurella strains not producing indole were investigated for their taxonomic relationships with Pasteurella multocida, Pasteurella haemolytica and Pasteurella canis. For all strains, DNA-DNA hybridization has revealed a high genetic relatedness at the species level to P. multocida and significantly lower homologies of only 18-41% towards P. haemolytica and 11-15% towards P. canis. Guanine plus cytosine values of 38.0 to 42.1 mol% and several phenotypic characters have been found to be different from the established pattern for P. multocida subspecies. It is suggested that the strains represent a new taxon, possibly another P. multocida subspecies.     

Specificity of DNA probes for the detection of toxigenic Pasteurella multocida subsp. multocida strains.

Five DNA probes directed against different regions of the gene that encodes the dermonecrotic toxin of Pasteurella multocida subsp. multocida were examined for their ability to identify toxigenic P. multocida subsp. multocida strains. The specificities of the probes were studied with 96 strains of P. multocida subsp. multocida and 22 strains of 11 other bacterial species. Results of colony hybridization assays using these probes indicated that two of the five probes have potential diagnostic value.     

Pasteurella multocida subsp. multocida and P. multocida subsp. septica Differentiation by PCR Fingerprinting and α-Glucosidase Activity

Pasteurella multocida is composed of three subspecies that are often differentiated by fermentation of sorbitol and dulcitol. We studied 35 dulcitol-negative P. multocida isolates from infected dog and cat bite wounds, 16 of which yielded weak and/or conflicting fermentation reactions in Andrades sorbitol, thus making it difficult to distinguish between the two dulcitol-negative subspecies of P. multocida, i.e., P. multocida subsp. multocida and P. multocida subsp. septica. All isolates and two control strains were further analyzed using a PCR fingerprinting technique with a single primer (M13 core) and assessed for alpha-glucosidase (alpha-Glu) activity. Although the PCR fingerprint patterns and alpha-Glu activity did not correlate well with the sorbitol fermentation reactions, they did correlate well with each other. All strains identified as P. multocida subsp. septica were positive for alpha-Glu activity and exhibited the group I PCR fingerprint profile. All strains categorized as P. multocida subsp. multocida displayed either the group II or group III PCR fingerprint profile; 9 of 11 of these isolates were alpha-Glu negative. These data suggest that both PCR fingerprinting and alpha-Glu activity provide reliable means for differentiating P. multocida subsp. multocida from P. multocida subsp. septica, particularly in strains that produce weak and/or discrepant sorbitol fermentation reactions.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to Pasteurella multocida in rabbits.

Three antigen preparations of Pasteurella multocida, lipopolysaccharide antigen, boiled-cell extract antigen, and boiled whole-bacterium antigen, were used in an enzyme-linked immunosorbent assay (ELISA) to detect rabbit immunoglobulin G antibody to P. multocida. The sensitivity of each antigen preparation was compared by using sera from P. multocida-infected and uninfected rabbits and sera from two rabbits immunized with different serotypes of P. multocida. In the ELISA, all three antigen preparations detected high titers of antibodies in infected rabbits and markedly lower levels in uninfected rabbits. When whole-bacterium or boiled-cell extract antigens were used, the ELISA detected antibodies in sera from both immunized rabbits, but with lipopolysaccharide antigen, only antibody to the homologous serotype was detected. Sera absorbed with P. multocida and Bordetella bronchiseptica, another respiratory pathogen of rabbits, revealed that antibodies detected in the ELISA did not cross-react. Since the lipopolysaccharide antigen was more difficult to prepare and may be type specific, and since the whole-bacterium antigen was the least sensitive, the boiled-cell extract was chosen as the best antigen preparation to use in the ELISA.     

Evaluation of transport media for Pasteurella multocida isolates from rabbit nasal specimens.

A suitable medium for the transport of Pasteurella multocida in nasal specimens from rabbits was investigated by using pure cultures of the organism and nasal swabs from infected rabbits. First, the ability of eight transport media to preserve the viabilities of P. multocida strains isolated from rabbits was studied. Cary-Blair medium and Leibovitz medium no. 15 (L-15) were found to be superior to the other six media tested, enabling survival of the organism for more than 14 days at room temperature. Second, the survival of P. multocida in nasal specimens was evaluated on both Cary-Blair medium and L-15. The recovery rate of the organism from these two media was more than 80 to 90% during 4 days of storage and decreased gradually with increasing preservation time. There were no significant differences (P > 0.05) in recovery rates of the organism between Cary-Blair medium and L-15. On the basis of these results, we recommend the use of Cary-Blair medium for the transport of P. multocida in rabbit nasal specimens because of the ease of transport of nasal swabs by mail.     

Pasteurella multocida produces a protein with homology to the P6 outer membrane protein of Haemophilus influenzae.

An antibody specific for a 16-kDa outer membrane protein of a rabbit strain of Pasteurella multocida was used to probe representatives of all 16 somatic serotypes of P. multocida, as well as the vaccine strains CU and M9, and all were shown to express the protein. The gene encoding this protein was cloned and sequenced and found to have extensive sequence homology with the gene encoding the P6 protein of Haemophilus influenzae. The protein in P. multocida has been designated P6-like. The gene encoding the P6-like protein was used to probe members of the family Pasteurellaceae and other gram-negative bacteria. Representatives of all 16 somatic serotypes (as well as the vaccine strains CU and M9) of P. multocida hybridized with the P6-like gene under conditions of high stringency. The DNA from H. influenzae hybridized weakly with the P6-like gene under these conditions, but Pasteurella haemolytica (representatives of A and T biotypes), Bordetella bronchiseptica, B. avium, Actinobacillus suis, A. suis-like, A. lignieresii, A. ureae, A. rossii, A. pleuropneumoniae, A. equuli, and various members of the family Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium) did not hybridize detectably. Under conditions of lower stringency, the P6-like gene also hybridized strongly with DNA from P. multocida, H. influenzae, and A. rossii but weakly with DNA from P. haemolytica and members of the genus Actinobacillus. These results suggest that the P6-like protein of P. multocida might be useful as an immunizing product to protect poultry from avian cholera. This suggestion stems from (i) our finding that the P6-like protein in P. multocida is widely distributed among all the somatic serotypes and (ii) the previous work of others demonstrating that the P6 protein of H. influenzae elicits a protective immune response in animal models of human disease.     

Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits.

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.     

Characterization of sucrose-negative Pasteurella multocida variants, including isolates from large-cat bite wounds

To validate the identification of Pasteurella multocida-like bacteria negative for acid formation from sucrose, including isolates from bite wounds caused by large cats, 17 strains were phenotypically and genotypically characterized. Phylogenetic analysis of partially sequenced rpoB and infB genes showed the monophyly of the strains characterized and the reference strains of P. multocida. The sucrose-negative strains formed two groups, one related to reference strains of P. multocida and the other related to a separate species-like group (taxon 45 of Bisgaard). DNA-DNA hybridization further documented the species-like nature of this group. Ribotyping showed the heterogeneity of all strains except four strains that shared the same ribotype and that were isolated from bovine lungs. Phylogenetic analysis by 16S rRNA sequence comparison showed the monophyly of the strains characterized and the reference strains of P. multocida. Two strains isolated from leopard bite wounds were related to the type strain of P. dagmatis; however, they represented a new taxon (taxon 46 of Bisgaard), in accordance with their distinct phenotypic and genotypic identifications. The present study documents that sucrose-negative strains of P. multocida-like bacteria belong to two genotypically distinct groups. The study further confirms the phenotypic heterogeneity of P. multocida strains and documents two new species-like taxa of Pasteurella related to P. multocida. Until diagnostic tools have been further elaborated, special care should be taken in the identification of Pasteurella-like bacteria isolated from bite wounds caused by large cats. The evidence of phenotypic and genotypic divergence calls for the further development of PCR tests and DNA sequencing to document doubtful isolates.     

Development and epidemiological applications of a bacteriophage typing system for typing Pasteurella multocida.

A bacteriophage typing system was developed for typing toxigenic and nontoxigenic strains of Pasteurella multocida. A phage set of 24 phages with different lytic spectra was isolated after mitomycin treatment of P. multocida strains, isolated mainly from pigs from herds with atrophic rhinitis. On a test set of 97 different strains isolated from pigs, these 24 phages were able to type 87% of the strains. The 97 test strains could be subdivided into 31 different types by reaction with the 24 phages. The reproducibility after subculture and storage of the strains was good (95%). Phage typing of 217 toxigenic P. multocida field isolates from 37 pig herds predominately with clinically atrophic rhinitis resulted in 18 different phage types and an overall typability of 68%. Of 24 herds from which more than three isolates of toxigenic P. multocida were obtained, a single phage type was demonstrated in 5 herds, while in 9 herds a single phage type represented at least half of the isolates. The phage types in the remaining 10 herds revealed no dominating phage type. The phage typing system described appears to be a valuable epidemiological tool for studying the spread of P. multocida.     

Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing Escherichia coli (STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes stx(1) and stx(2), respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (n = 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n = 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for stx(1) and/or stx(2). Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.