Comparison of DNA fingerprinting and serotyping for identification of avian Pasteurella multocida isolates.

The DNA fingerprint profiles and serotypes of 63 avian Pasteurella multocida field isolates, 13 attenuated vaccine isolates (propagated from vaccines manufactured by five companies), and 16 somatic reference strains were compared. DNA fingerprinting established the relationship of isolates that could not be distinguished by serotyping. Of the 76 isolates, 28 DNA fingerprint profiles and 12 somatic types were recognized. One isolate was nonreactive with 16 reference somatic and 5 reference capsule-type antisera. Thirty-one field isolates and seven vaccine isolates were identified as capsule type A. Twenty-nine field isolates and six vaccine isolates were nonencapsulated. Three field isolates were capsule type F. Isolates of capsule types B, D, and E were not found. One field isolate, identified as somatic type 7, had a DNA fingerprint identical to that of the somatic reference type 6 profile. Twelve field isolates had profiles identical to the somatic reference type 3 strain profile; 11 of these were identified as somatic type 3, 4, and 1 was identified as somatic type 3. The DNA fingerprint profiles of 50 field isolates and 13 attenuated vaccine isolates did not match profiles of the 16 somatic type reference strains. Twenty-five DNA fingerprint profiles were recognized from 30 of these field isolates. The DNA fingerprint profiles of 20 field isolates and 13 attenuated vaccine isolates were identical. Three somatic types (3; 3,4; and 4,16) were identified from the field isolates, and two somatic types (3 and 3,4) were identified from the attenuated vaccine isolates. DNA fingerprinting is useful for accurate identification and epidemiologic study of P. multocida isolates.     

Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system

Current serotyping methods classify Pasteurella multocida into five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci of P. multocida A:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289-296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types.     

Random amplified polymorphic DNA and amplified fragment length polymorphism analyses of Pasteurella multocida isolates from fatal fowl cholera infections

Fowl cholera, a disease caused by Pasteurella multocida, continues to be a major problem for the poultry industry. The sources of pathogenic organisms responsible for most sporadic epidemics remain unconfirmed, although attenuated vaccines that retain a low level of virulence have occasionally been implicated in outbreaks of the disease. One of the vaccines most commonly used to prevent fowl cholera is the M-9 strain. In the present study, 61 clinical isolates from turkeys that died of fowl cholera from 1997 to 1999 on 36 Utah farms were analyzed and compared to the M-9 vaccine strain. Genetic analyses of the isolates were done by random amplified polymorphic DNA (RAPD) analysis and amplified fragment length polymorphism (AFLP) fingerprinting. The results of these genetic analyses were correlated with the vaccination status of the flock, isolate serotype, and geographic location. Although both genetic techniques effectively identified similar subtle genomic differences, RAPD analysis provided only 77% of the detail provided by AFLP analysis. While a relationship between genetic profile and serotype was evident, no significant relationship indicating geographic influence was found (P = 0.351). Interestingly, organisms isolated from vaccinated flocks were significantly closer genetically to the M-9 vaccine strain than isolates from unvaccinated birds were (P = 0.020). Statistical analyses revealed that this relationship could not have been determined by serotyping alone (P = 0.320), demonstrating the value of AFLP and RAPD analyses in the characterization of disease-causing strains.     

Antigenically related iron-regulated outer membrane proteins produced by different somatic serotypes of Pasteurella multocida.

An 84-kilodalton outer membrane protein was expressed when Pasteurella multocida, somatic serotype 3, was grown in brain-heart infusion broth containing the iron chelator dipyridyl but not in brain-heart infusion broth alone. Antigenically related outer membrane proteins of various molecular masses were also expressed by P. multocida strains belonging to all of the other 15 somatic serotypes (somatic serotype 12 being the possible exception) as well as by isolates expressing somatic antigens representative of multiple somatic serotypes when grown under the same conditions of iron deprivation.     

Typing of Pasteurella multocida isolated from pigs with and without porcine dermatitis and nephropathy syndrome

Porcine dermatitis and nephropathy syndrome (PDNS) is a sporadic, usually fatal disease of growing and finishing pigs that has been recognized in many pig-producing countries. Pasteurella multocida strains isolated from 15 pigs with PDNS and 51 pigs without PDNS were characterized by capsule and somatic antigen typing, random amplified polymorphic DNA (RAP-D) typing, and restriction analysis of genomic DNA using pulsed-field gel electrophoresis (PFGE). While capsular, somatic, and RAP-D typing did not discriminate PDNS isolates from non-PDNS isolates, all of the isolates from PDNS cases showed an identical ApaI PFGE restriction pattern. This pattern was also found in a high proportion (36%) of P. multocida strains isolated from non-PDNS cases. Isolation of a single variant of P. multocida from tissues of pigs with PDNS warrants further investigation into the possible role of these bacteria in the etiology of the disease.     

Distribution of a monoclonal antibody-recognized protective protein immunogen on the outer membranes of Pasteurella multocida rabbit isolates.

The distribution of a monoclonal antibody (MAb)-recognized protective protein immunogen on the outer membrane of 153 Pasteurella multocida rabbit isolates was determined by dot blot (DB) analysis. MAb 1608 reacted with 36 (24%) of the 153 clinical isolates. The DB-positive clinical isolates expressed capsular antigens A, D, and nontypable and somatic antigens 2, 3, 10, 12, 15, and nontypable. Western blot (immunoblot) analysis with adsorbed and eluted MAb 1608 confirmed that the antigenic determinant identified was located on the cell surface. With MAb 1608 as a probe for antibody-accessible radioimmunoassay, 31 of 36 DB-positive P. multocida rabbit isolates were shown to have surface-exposed and antibody-accessible antigenic determinants, while 44 of 44 DB-negative isolates were negative by antibody-accessible radioimmunoassay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed DB-negative P. multocida isolates both with (6 of 13, 46%) and without (7 of 13, 54%) the 37.5-kilodalton protein. This study establishes that the protective antigenic determinant of the 37.5-kilodalton outer membrane protein is present in 24% of rabbit clinical isolates tested and is detectable in P. multocida strains distributed among the major somatic types (3, 10, 12, and 15) and the capsular types (A and D) commonly isolated from rabbits in North America.     

Genomic DNA restriction site heterogeneity in bovine Pasteurella multocida serogroup A isolates detected with an rRNA probe.

A total of 81 Pasteurella multocida isolates from healthy and diseased dairy and beef cattle originating from various geographical locations was examined by rRNA gene restriction site polymorphism analysis (ribotyping), restriction endonuclease analysis (REA), SDS-PAGE analysis of whole-cell (WCP) and outer-membrane (OMP) proteins, and capsule and somatic serotyping. Bacterial strains were isolated from nose, lung and in one case testicle, of Holstein and cross-bred beef cattle. The isolates represented for the most part serogroup A3 (88%). Ribotyping was performed on DNA digested with HaeII, electrophoresed and then hybridised with 32P-labelled 16S-23S rRNA from Escherichia coli. Six ribotypes (R1-R6) and 10 REA types were found among the 81 isolates with similar discrimination index (DI) of c. 0.60. Protein profiles revealed reproducibility and high levels of polymorphisms among lung isolates. Isolates were compared according to their geographical habitat, their isolation from dairy or from beef cattle and from nasal cavities or lungs. No correlation was apparent between geographical locations and ribotypes. Overall, isolates obtained from dairy cattle were predominantly R1, whereas those obtained from beef cattle were equally distributed between R1 and R2. R1 was more representative of lung isolates. For some strains, particularly the single isolate ribotypes, good correlation was achieved between WCP analysis, REA types and ribotypes. For others, REA to some extent and WCP profiles were able to discriminate among isolates within ribotypes. The data suggest that a combination of ribotyping, REA and WCP analysis is useful for investigating the epidemiology of bovine P. multocida serogroup A.     

Comparison of PCR-based methods for typing Escherichia coli

Objective  To establish a library typing system appropriate for studying cross-transmission of Escherichia coli.
Methods  Eighteen epidemiologically unrelated isolates were genotyped by means of pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), repetitive (rep) PCR, and fluorescent amplified fragment length polymorphism (AFLP). Fingerprints were analyzed either by Pearson correlation or, in the case of AFLP, by Dice coefficients employing the novel 'uncertain band' software tool from GelCompar II. During a nine-month period, 112 isolates taken from 93 patients hospitalized in five intensive care units were analyzed by use of the two most discriminative PCR typing methods.
Results  Genotyping by RAPD and rep-PCR revealed insufficient discrimination. Among 18 epidemiologically unrelated strains with 17 different PFGE patterns, IS3 rep-PCR and AFLP distinguished 10 and 18 types, respectively. Comparison of the different methods for analysis of AFLP fingerprints showed that the Dice coefficients, which ignore 'uncertain bands', offered the best concordance with visual interpretation. Consecutive isolates originating from the same patient differed in less than three fragments.
Conclusions  AFLP analysis showed the highest discriminative capacity for PCR typing of E. coli isolates. Analysis of fingerprints employing the Dice coefficients proved the most efficient method for an automated software-based retrieval of visually indistinguishable genotypes in an AFLP fingerprint database.     

Characterization of Pasteurella multocida from nasal cavities of piglets from farms with or without atrophic rhinitis.

A total of 137 strains of Pasteurella multocida isolated from the nasal tracts of pigs with and without clinical atrophic rhinitis (AR) were studied for their biochemical, antigenic, and toxigenic characteristics. There were no major biochemical differences among the P. multocida isolates. Capsular antigen types A and D were both present in the nasal cavities of the pigs with or without clinical AR. However, the prevalence of type D was higher on farms with pigs with AR. Types A and D with different somatic antigens could both be present in the same pig. There was no correlation between somatic types and/or capsular types with the clinical AR status of the pigs on the farm. Toxigenic isolates were found only in pigs which had a problem of clinical AR, and a great majority of these isolates belonged to type D. Since there was a high level of heterogeneity of the strains in the P. multocida population on a farm, several strains should be characterized before the diagnosis of AR could be excluded on the basis of the absence of isolation of rhinopathogenic P. multocida strains.     

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.     

Molecular fingerprinting of Legionella species by repetitive element PCR.

Repetitive element PCR (rep-PCR) uses outward-facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphisms of rep-PCR amplification products can serve as strain-specific molecular fingerprints. Primers directed at the repetitive extragenic palindromic element were used to characterize isolates of Legionella pneumophila and other Legionella species. Substantial variation was seen among the rep-PCR fingerprints of different Legionella species and serogroups. More limited, but distinct, polymorphisms of the rep-PCR fingerprint were evident among epidemiologically unrelated isolates of L. pneumophila serogroup 1. Previously characterized Legionella isolates from nosocomial outbreaks were correctly clustered by this method. These results suggest the presence of repetitive extragenic palindromic-like elements within the genomes of members of the family Legionellaceae that can be used to discriminate between strains within a serogroup of L. pneumophila and between different Legionella species. rep-PCR appears to be a useful technique for the molecular fingerprinting of Legionella species.     

Direct PCR analysis for toxigenic Pasteurella multocida.

A more rapid, accurate method to detect toxigenic Pasteurella multocida is needed for improved clinical diagnosis, farm biosecurity, and epidemiological studies. Toxigenic and nontoxigenic P. multocida isolates cannot be differentiated by morphology or standard biochemical reactions. The feasibility of using PCR for accurate, rapid detection of toxigenic P. multocida from swabs was investigated. A PCR protocol which results in amplification of an 846-nucleotide segment of the toxA gene was developed. The PCR amplification protocol is specific for toxigenic P. multocida and can detect fewer than 100 bacteria. There was concordance of PCR results with (i) detection of toxA gene with colony blot hybridization, (ii) detection of ToxA protein with colony immunoblot analysis, and (iii) lethal toxicity of sonicate in mice in a test set of 40 swine diagnostic isolates. Results of an enzyme-linked immunosorbent assay for ToxA agreed with the other assays except for a negative reaction in one of the 19 isolates that the other assays identified as toxigenic. In addition to accuracy, as required for a rapid direct specimen assay, toxigenic P. multocida was recovered efficiently from inoculated swabs without inhibition of the PCR. The results show that PCR detection of toxigenic P. multocida directly from clinical swab specimens should be feasible.     

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.     

Comparison of indirect hemagglutination and rapid plate agglutination tests with counterimmunoelectrophoresis for typing Pasteurella haemolytica.

A rapid, simple, and accurate counterimmunoelectrophoresis (CIE) technique was developed and compared with the indirect hemagglutination and rapid plate agglutination techniques for serotyping cultures of Pasteurella haemolytica. The CIE test had 100% correlation with the conventional indirect hemagglutination test and, after serum absorption, correctly identified cultures representing the 12 established serotypes and 49 field isolates of P. haemolytica with reasonable rapidity. Cross-reactions were observed in the CIE and rapid plate agglutination tests but not in the indirect hemagglutination test with antisera prepared from the 12 established serotypes. These cross-reactions were eliminated from the CIE test but not from the rapid plate agglutination test by absorption of antisera with cells which possessed the cross-reacting antigens. Avian isolates of P. haemolytica did not type with antisera to the 12 established serotypes by any of the methods. Both homologous and heterologous reactions were observed with these strains in the rapid plate agglutination and CIE tests with antisera prepared from six selected cultures. These results support the previous finding that the taxonomic relationship of these avian strains to P. haemolytica is questionable.     

O-serotyping Providencia alcalifaciens.

The O-serotyping scheme for Providencia was tested on Providencia alcalifaciens isolates collected mostly from two hospitals. The specificites of the somatic (O) antigens of P. alcalifaciens were found to be different from those of Providencia stuartii, and separation of the Providencia typing scheme to allow separate typing of each species led to more efficient typing. All but 4 of 86 isolates were typable. Eighteen serotypes occurred among 53 typable isolates obtained from a pediatric hospital, and 11 occurred among 19 isolates from a general hospital. Thirty-two percent of the isolates from the pediatric hospital belonged to serotype O3, the most frequently isolated and most widely distributed type. The use of the serotyping scheme for P. alcalifaciens is advocated for further studies to examine strains of the species for enteropathogenic types.     

Comparison of an automated repetitive sequence-based PCR microbial typing system to pulsed-field gel electrophoresis for analysis of outbreaks of methicillin-resistant Staphylococcus aureus

Rapid and sensitive methods for accurate strain delineation are essential for monitoring and preventing transmission of methicillin-resistant Staphylococcus aureus (MRSA). Pulsed-field gel electrophoresis (PFGE) has been the standard technique for strain typing most bacterial species including MRSA. The goal of this study was to compare the performance of the DiversiLab microbial typing system (Bacterial BarCodes, Inc., Houston, TX) (rep-PCR) to that of PFGE for typing MRSA isolates from five well-defined outbreaks. The DiversiLab rep-PCR assay is a rapid, semiautomated method based on PCR amplification of specific regions between noncoding repetitive sequences in the bacterial genome. rep-PCR was performed according to the manufacturer's recommendations, and the results were analyzed and dendrograms were generated using the DiversiLab analysis software (version 2.1.66 a). PFGE was performed and interpreted according to published procedures. rep-PCR results using similarity indices (SI) of 80%, 85%, and 90% were compared to PFGE analysis. In addition, intra- and interrun reproducibility was determined for rep-PCR. Overall, correct assignment to outbreak versus nonoutbreak clusters occurred for 91 of 109 isolates (85% agreement) when using a SI of 85%. For each specific outbreak, concordance between rep-PCR and PFGE ranged from 73% to 100%. There were 18 discrepant results (17%). Fourteen isolates were unique by PFGE, but they were placed in clusters by rep-PCR; the other 4 were placed in clusters different from those assigned by PFGE. Intra- and interrun reproducibility was excellent. Times to results were 12 to 24 h for rep-PCR compared to 2 to 4 days for PFGE. Rapid, standardized results and excellent reproducibility make rep-PCR a valuable tool for use in MRSA investigations. However, since rep-PCR was less discriminatory than PFGE, we recommend that it be used to screen isolates, followed by testing isolates which share the same rep-PCR pattern with a more sensitive method, such as PFGE or multilocus sequence typing.     

Avian pasteurellosis: Taxonomy of the organisms involved and aspects of pathogenesis.

The taxonomy of the family Pasteurellaceae Pohl 1981 appears to be as complex as that of Enterobacteriaceae. 16S rRNA sequencing indicates that the family should be divided into more than 20 genera. According to phylogenetic investigations, the genus Pasteurella sensu stricto includes three subclusters, two of which represent taxa mainly associated with avian hosts. True species of the genera Actinobacillus and Haemophilus have not been reported from birds. Several new taxa, which have been shown to belong to the family Pasteurellaceae Pohl 1981 have been reported from birds. Some of these seem to represent genus-like structures. Due to a high degree of host-specificity observed for many taxa belonging to the family, the existence of many more species can be foreseen as more avian species are examined. The pathogenesis of Pasteurella infections in birds is poorly understood. However, it has long been recognized that the severity of the disease and its incidence may vary considerably depending on several factors associated with the host, the environment or the bacterial strain. Several virulence factors of P. multocida, may be of importance for infection of birds and are discussed. There is some evidence that the capsule of P. multocida is of importance in the protection against phagocytosis by immunocompetent cells and that it mediates resistance to complement. Endotoxin is another factor which has persistently been associated with pathogenicity of P. multocida. There are some indications that the P. multocida exotoxin (PMT), which is involved in the pathogenesis of atrophic rhinitis in swine, may play a role in some avian infections. High molecular weight outer membrane proteins of P. multocida have been speculated to be the main iron acquisition system of avian strains of P. multocida and thereby representing virulence factors. An outer membrane protein with anti-phagocytic activity has also been demonstrated. The potential role of different enzymes in the pathogenesis P. multocida infections has been investigated, but not conclusively demonstrated. Apart from the knowledge concerning the PMT encoding gene toxA, very little is known about the genetic basis of diseases caused by pasteurellas associated with avian species.     

Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds.

One hundred and sixty-four clinical isolates of Pasteurella multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTP-labeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.     

Hemagglutination by Pasteurella multocida of porcine origin.

A total of 25 fresh isolates of Pasteurella multocida from pigs were tested for the ability to agglutinate erythrocytes of different origins. Of the 18 isolates from pigs with atrophic rhinitis (AR), 8 (44%) agglutinated human type O erythrocytes, whereas none of the 7 isolates from pigs without AR did so. Hemagglutination was mannose sensitive, and the activity was destroyed by heating and by trypsinization but was not affected by formaldehyde treatment or by homogenization of bacterial cells. The other 14 erythrocyte species tested, including human types A and B, were not agglutinated by P. multocida. The present study provides evidence that certain P. multocida isolates from pigs with AR possess a mannose-sensitive hemagglutinin.