Identification and typing of Pasteurella multocida: a review

Pasteurella multocida is an important pathogen of many avian species. This review critically examines recent developments in new-generation tests for the identification and typing of this bacterium. Two polymerase chain reaction (PCR) tests have been reported for P. multocida. Both tests show promise as diagnostic tests that could be considered for routine use. However, there have not yet been effective evaluation studies that examine the ability of these new tests to distinguish between P. multocida, both typical and atypical isolates, and the range of other P. multocida-like organisms found in avian species. One PCR, reported by Townsend et al. (Journal of Clinical Microbiology, 36, 1096-1100), has been the more fully evaluated and is the better choice, at this stage, for laboratories considering the use of PCR technology for detection of P. multocida. An important point is that the PCR tests have been validated by use on pure cultures or enrichment broths - not on direct examination of body tissues. To date, there have been five different technologies used to type avian P. multocida: restriction endonuclease analysis (REA), ribotyping, pulsed field gel electrophoresis, repetitive extragenic palindromic-PCR (REP-PCR) and multi-locus enzyme electrophoresis (MLEE). The methodology underlying these techniques is briefly explained and the performance of these techniques with regards to the typing of avian P. multocida is critically examined. For smaller laboratories that are investigating outbreaks of fowl cholera, it would appear that REA and REP-PCR are the typing methods of choice. For central reference laboratories that are considering studies of large collections of isolates, MLEE supported by two of the other methods would appear the most suitable techniques

Identification and typing of Pasteurella multocida: A review

Blackall and Miflin critically examine recent developments in new-generation tests for the identification and typing of Pasteurella multocida. Two polymerase chain reaction (PCR) tests have been reported for P. multocida as both show promise as diagnostic tests that could be considered for routine use

Development of a 23S rRNA-based PCR assay for the identification of Pasteurella multocida

Aims: The aim of this work was to develop a rapid diagnostic test for Pasteurella multocida. Methods and Results: A polymerase chain reaction (PCR) assay using primers derived from the 23S rRNA gene sequence of Past. multocida was developed. The PCR assay correctly identified all 144 isolates of Past. multocida tested, including type strains of the three subspecies as well as the reference strains for the Heddleston and Carter typing schemes. Of 20 closely related bacteria from the family Pasteurellaceae tested, only the type strains of Past. canis biovar 2 and Past. avium biovar 2 were positive. These two bacteria, formerly known as Bisgaard Taxon 13, are the closest phylogenetic relatives of Past. multocida based on 16S ribosomal rRNA. All phylogenetically unrelated avian and porcine organisms tested were negative. Conclusions: This PCR enables rapid identification of Past. multocida colonies from avian or porcine origin. Significance and Impact of the Study: Veterinary diagnostic laboratories can use this PCR to rapidly and accurately diagnose fowl cholera and porcine pasteurellosis

Molecular characterization of isolates of Haemophilus paragallinarum from China by ribotyping

Ribotyping profiles were determined for 12 Page serovar A isolates of H. paragallinarum from 5 outbreaks of infectious coryza in commercial layer flocks in Hebei province in China between 1986 and 1993. Four enzymes were used to generate restriction digests of chromosomal DNA: Hae III, Hin dIII, Hpa II and Ssp I. The digests were probed with a digoxigenin-11-dUTP-labelled DNA probe produced by PCR amplification of the 16S rDNA of the type strain of H. paragallinarum . Only one profile was seen among the 12 isolates when using either Hin dIII or Ssp I. Three different ribotype profiles were seen with Hpa II, while 4 different profiles were detected with Hae III. Cluster analysis of the profiles generated by Hpa II and Hae III corresponded with the known epidemiological history of the isolates. This appears to be the first report on molecular characterization of isolates of H. paragallinarum from China

Development and application of DNA probes and PCR tests for Haemophilus paragallinarum

Screening a genomic DNA library of H. paragallinarum strain Modesto identified 4 clones that reacted specifically with all 56 isolates of H. paragallinarum tested and failed to react with 24 closely related bacteria from the genera Pasteurella and Actinobacillus . All 4 clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae . The probes based on these 4 clones were approximately 1.8, 2.3, 3.5, and 5.5 kb in size. The 4 probes detected between 7.8 and 31.25 ng of purified DNA from homologous strains with no obvious correlation between probe size and sensitivity. The smallest probe, termed P601, was partially sequenced, and primers for 2 polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were specific, all 41 isolates of H. paragallinarum tested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR rests were able to detect 1 pg DNA and between 102 and 103 cells. A method for using the HPG-2 PCR test directly on sinus swabs was developed. Using this method, there was 100% agreement between culture and the direct HPG-2 PCR for the 36 swabs processed. The DNA probes and PCR tests appear to be useful diagnostic tools for the detection of infectious coryza. The tests can be used as confirmation following the isolation of a haemophilic organism. The HPG-2 PCR test also appears to be a potential alternative to culture

Development and application of DNA probes and PCR tests for Haemophilus paragallinarum

Screening a genomic DNA library of H. paragallinarum strain Modesto identified 4 clones that reacted specifically with all 56 isolates of H. paragallinarum tested and failed to react with 24 closely related bacteria from the genera Pasteurella and Actinobacillus . All 4 clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae . The probes based on these 4 clones were approximately 1.8, 2.3, 3.5, and 5.5 kb in size. The 4 probes detected between 7.8 and 31.25 ng of purified DNA from homologous strains with no obvious correlation between probe size and sensitivity. The smallest probe, termed P601, was partially sequenced, and primers for 2 polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were specific, all 41 isolates of H. paragallinarum tested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR rests were able to detect 1 pg DNA and between 102 and 103 cells. A method for using the HPG-2 PCR test directly on sinus swabs was developed. Using this method, there was 100% agreement between culture and the direct HPG-2 PCR for the 36 swabs processed. The DNA probes and PCR tests appear to be useful diagnostic tools for the detection of infectious coryza. The tests can be used as confirmation following the isolation of a haemophilic organism. The HPG-2 PCR test also appears to be a potential alternative to culture

Confirmation that PCR can be used to identify NAD-dependent and NAD-independent Haemophilus paragallinarum isolates

75 bacteria tentatively identified as H. paragallinarum, 8 identified as Ornithobacterium rhinotracheale and 13 identified as nicotinamide adenine dinucleotide (NAD)-independent Pasteurella spp. were isolated from chickens with respiratory infection in various provinces in South Africa. The isolates were characterized by conventional biochemical and serological methods. A polymerase chain reaction (PCR) assay specific for H. paragallinarum was used to identify the cultures directly from colonies. The PCR assay gave positive results for all isolates that were identified by conventional methods as H. paragallinarum, irrespective of whether they were NAD-dependent (43 isolates) or NAD-independent (32 isolates). The 8 isolates that were identified by conventional methods as O. rhinotracheale and the 13 isolates identified as various Pasteurella spp. gave negative results in the PCR assay. It is concluded that colony PCR is a rapid method for uniquely identifying NAD-dependent and NAD-independent strains of H. paragallinarum and distinguishing them from other bacteria, such as O. rhinotracheale and Pasteurella spp

Confirmation that PCR can be used to identify NAD-dependent and NAD-independent Haemophilus paragallinarum isolates

75 bacteria tentatively identified as H. paragallinarum, 8 identified as Ornithobacterium rhinotracheale and 13 identified as nicotinamide adenine dinucleotide (NAD)-independent Pasteurella spp. were isolated from chickens with respiratory infection in various provinces in South Africa. The isolates were characterized by conventional biochemical and serological methods. A polymerase chain reaction (PCR) assay specific for H. paragallinarum was used to identify the cultures directly from colonies. The PCR assay gave positive results for all isolates that were identified by conventional methods as H. paragallinarum, irrespective of whether they were NAD-dependent (43 isolates) or NAD-independent (32 isolates). The 8 isolates that were identified by conventional methods as O. rhinotracheale and the 13 isolates identified as various Pasteurella spp. gave negative results in the PCR assay. It is concluded that colony PCR is a rapid method for uniquely identifying NAD-dependent and NAD-independent strains of H. paragallinarum and distinguishing them from other bacteria, such as O. rhinotracheale and Pasteurella spp