J.K. Miflin

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

A genomic DNA library of Haemophilus paragallinarum strain Modesto was created. Screening of this library identified four 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 four clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae. The probes based on these four clones were approximately 1.8, 2.3, 3.5, and 5.5 kb in size. The four probes were able to detect 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 two polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were shown to be specific, all 41 isolates of H. paragallinarum tested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR tests were able to detect 1 pg DNA and between 10(2) and 10(3) 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 confirmatory tests following the isolation of a hemophilic organism. As well, the HPG-2 PCR test appears to be a potential alternative to culture.

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.

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.

Survival of Campylobacter spp. in Darkling Beetles (Alphitobius diaperinus) and Their Larvae in Australia

ABSTRACT Campylobacter infection is the most frequently reported notifiable food-borne disease in humans in Australia. Our studies investigated the persistence of Campylobacter spp. in or on darkling beetles (Alphitobius diaperinus) and their larvae. Our results in analyses with chickens confirm that, unless very short turnaround times are used (<72 h), beetles colonized in one production cycle (i.e., one batch of chickens) are most unlikely to still be colonized during the next cycle of chickens.

Phenotypic and molecular characterization of V-factor (NAD)-independent Haemophilus paragallinarum

In South Africa from early 1989 onward, strains of Haemophilus paragallinarum not requiring nicotinamide adenine dinucleotide (NAD) have been isolated from commercial chickens suffering from infectious coryza. Fifteen of these field isolates were characterized by biochemical typing, serotyping, restriction endonuclease analysis (REA), and ribotyping. The chosen isolates represented diversity in geographic location, time of disease outbreak, and type of flock. All were typical of the species in biochemical properties, except that they were NAD-independent, and all were Page serovar A. REA was performed with three enzymes: HindIII, HpaII, and SspI. All isolates gave identical REA profiles with all three enzymes. Ribotyping was performed using a probe that consisted of the plasmid pUC19 into which the 16S rRNA operon of H. paragallinarum had been inserted. All 15 isolates gave the same ribotyping profile using each of the three enzymes. As a group, the NAD-independent strains gave REA profiles and ribotypes that were very different from a range of classic South African strains isolated before 1989. Our results strongly suggest that the NAD-independent isolates are clonal in nature.

Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

Molecular characterization of isolates of Haemophilus paragallinarum from China by ribotyping

Ribotyping profiles were determined for 12 Page serovar A isolates of Haemophilus paragallinarum from five outbreaks of infectious coryza in Hebei province in the Peoples Republic of China. Four enzymes were used to generate restriction digests of chromosomal DNA: HaeIII, HindIII, HpaII and SspI. 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 HindIII or SspI. Three different ribotype profiles were seen with HpaII, while four different profiles were detected with HaeIII. Cluster analysis of the profiles generated by HpaII and HaeIII corresponded with the known epidemiological history of the isolates. These results are the first molecular characterization of isolates of H. paragallinarum from China and confirm the existence of genetic diversity in the H. paragallinarum population in that country.