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P J Coloe

Publications and source records attributed to P J Coloe.

At least 19 recordsLinked to original sources
PubMed2001

Specific identification, grouping and differentiation of Campylobacter jejuni among thermophilic campylobacters using multiplex PCR.

Campylobacter species such as C. jejuni and C. coli are recognized as major causes of acute gastroenteritis world-wide. Although C. jejuni and C. coli are usually non-pathogenic in birds and animals, they cause enteric disease in humans and the source of infection is often the consumption of contaminated foodstuffs. In this paper we report on the development and use of a multiplex PCR of C. jejuni genomic DNA which yielded a PCR product with a unique polymorphic site that can be used to quickly and accurately identify and group C. Jejuni isolates from any source including DNA, cell culture, skin washings and faecal samples. The test is simple and sensitive and can detect purified DNA from a single bacterium 10(2) cells from crude lysates, 10(3) cells in seeded faeces and 120 cells/ml of washing of 1 cm2 skin fragments of chicken skin.

Animals↗
PubMed2001

Differentiation between Campylobacter hyoilei and Campylobater coli using genotypic and phenotypic analyses.

Genotypic and phenotypic methods were applied to investigate differences between the closely related species Campylobacter hyoilei and Campylobacter coli. A unique DNA sequence from C. hyoilei was used to design a specific PCR assay that amplified a DNA product of 383 bp for all C. hyoilei strains, but not other Campylobacter species, including C. coli. The PCR assay could detect 100 fg pure C. hyoilei DNA, 2 x 10(2) c.f.u. ml(-1) using cultured cells and 8.3 x 10(3) c.f.u. 0.1 g(-1) in faeces. The C. hyoilei sequence utilized for specific detection and identification of this species showed similarities to sequences from bacteriophages Mu, P2 and 186, suggesting lysogination of the ancestral C. hyoilei genome. Activities of a set of 15 enzymes that participate in a variety of cellular functions, including biosynthesis, catabolism, energy generation, maintenance of redox balance and phosphate utilization, were tested using sets of strains of C. hyoilei and C. coli. Comparison of mean rates of enzyme activities revealed significant differences between species in the values determined for seven of these activities. Both the genetic and phenotypic data indicate that C. hyoilei is a unique Campylobacter species.

Bacteriophage P2↗
PubMed2000

Identification of a Salmonella typhimurium genomic region involved in invasion of HeLa and Henle-407 epithelial cells.

To identify the invasion determinant, a cosmid library was constructed by cloning a genomic library of Salmonella typhimurium 82/6915 into a cosmid vector, pLA2917. A genomic region involved in invasion of cultured HeLa and Henle-407 cells was subcloned into plasmid pGEM-7Z. E. coli strain DH1 carrying pSV6235 consisting of a S. typhimurium 4.6 kb genomic region in pGEM-7Z showed invasion of cultured HeLa and Henle-407 cells. Nested sequential deletions were introduced into the 4.6 kb genomic region of pSV6235. The E. coli recombinants which contained less than 1.5 kb deletions from the 5' end (SmaI site) of the genomic region invaded the cells as effectively as DH1 (pSV6235). The invasion of the recombinants carrying over 2.0 kb deletions from the end of pSV6235 was significantly inactivated compared to DH1 (pSV6235). Restriction enzyme analysis showed that the 3.1 kb fragment from the 3' end of the 4.6 kb genomic region was distinguished from the Salmonella pathogenicity I genes of S. typhimurium such as the inv, spa, and hil regions showing invasion of the cultured eukaryotic cells.

Cell Line↗
PubMed2000

Detection of a novel campylobacter cytotoxin.

The culture filtrates from 10 Campylobacter species were screened for the presence of cytotoxins on a variety of selected tissue culture cell lines. Some Campylobacter jejuni strains showed no effects on tissue culture cell lines compared with other C. jejuni strains, especially C. jejuni 81116, which consistently produced a cytotoxin that was lethal to tissue culture cells. It was observed that CHO cells were the most sensitive cell line in detecting campylobacter cytotoxins. Samples containing the culture filtrate of C. jejuni 81116 prepared at various growth stages were used to determine the subcellular location of the cytotoxin. This C. jejuni 81116 cytotoxin appears to be a heat-stable toxin that is secreted from the cell during stationary phase; cytotoxin activity can be abolished with proteolytic enzymes.

Animals↗
PubMed2000

The galE gene of Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence.

Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from Campylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complemented galE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-type Salmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuni encoded this epimerase. We constructed a C. jejuni galE mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galE gene in the synthesis of C. jejuni LPS. The galE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

Amino Acid Sequence↗
PubMed2000

Genetic characterization of Campylobacter jejuni O:41 isolates in relation with Guillain-Barré syndrome.

Campylobacter jejuni O:41 strains are found in association with Guillain-Barré syndrome in South Africa. Strains of this serotype collected over 17 years were characterized by amplified fragment length polymorphism and flagellin typing to determine their clonal nature. Despite minor variation in GM1 expression, all of the strains were genetically indistinguishable, indicating that they are representative of a genetically stable clone.

Campylobacter Infections↗
PubMed1999

Bacterial expression of the major antigenic regions of porcine rotavirus VP7 induces a neutralizing immune response in mice.

The outer capsid protein of rotavirus, VP7, is a major neutralization antigen. A chimeric protein comprising Escherichia coli (E. coli) outer membrane protein A (OmpA) and part of porcine rotavirus VP7 containing all three antigenic regions (217 amino acids) was expressed in Salmonella and E. coli as an outer-membrane associated protein. Mice immunized intraperitoneally or orally, respectively, with live E. coli or Salmonella cells expressing this chimeric protein produced antibodies against native VP7 as determined by enzyme-linked immunosorbent assays and neutralization tests. This indicates that the VP7 fragment from a porcine rotavirus which is antigenically similar to human rotavirus serotype 3, when expressed in bacteria as a chimeric protein, can form a structure resembling its native form at least in some of the major neutralization domains. These results indicate that the use of a live bacterial vector expressing rotavirus VP7 may represent a strategy for the development of vaccines against rotavirus-induced diarrhoea in infants.

Animals↗
PubMed1999

Bacteriological and molecular analysis of rifampin-resistant Mycobacterium tuberculosis strains isolated in Australia.

To develop a better understanding of the epidemiology and molecular biology of rifampin-resistant Mycobacterium tuberculosis strains in Australia, 50 clinical isolates (33 rifampin-resistant and 17 rifampin-sensitive strains) cultured between 1990 and 1997 were analyzed by a number of bacteriological and molecular techniques. Examination of the drug resistance profiles of the 33 rifampin-resistant isolates revealed that 91% were resistant to rifampin in combination with resistance to isoniazid, 88% were resistant to rifampin on first isolation, and 81% showed cross-resistance with rifabutin. On the basis of the demographic data provided for the patients infected with the rifampin-resistant strains, 90% of the patients were born overseas. Of these patients, 64% developed clinical symptoms within 5 years of residence in Australia. On a molecular level, analysis of the rpoB gene revealed that 97% of the rifampin-resistant isolates had missense mutations within a conserved region of the gene, and eight types of missense mutations were detected. Of the 31 rifampin-resistant isolates that were typed by restriction fragment length polymorphism (RFLP) analysis, 28 distinct patterns were obtained by RFLP analysis with IS6110, and three clusters of genetically related isolates were identified. All isolates within the clusters were from patients who were born overseas and who had the same country of origin. The results from this study provide an overview of the current situation of rifampin resistance in Australia and can serve as a basis for continued monitoring of drug-resistant M. tuberculosis strains isolated within the country.

Adolescent↗
PubMed1998

Isolation and molecular analysis of colonising and non-colonising strains of Campylobacter jejuni and Campylobacter coli following experimental infection of young chickens.

Fourteen-day-old chickens were inoculated with selected Campylobacter coli and C. jejuni strains. C. jejuni strains were of two subgroups based on a polymorphism detected using a DNA probe and represented the profiles typical for the majority of strains of either chicken or human origin. All C. coli strains previously isolated from humans colonised chickens, whereas from 4/7 C. jejuni strains of human origin, failed to colonise. Of 12 Campylobacter strains of chicken origin, 10 established a persistent colonisation in the chickens, and 2 strains colonised poorly or not at all. Four strains that failed to colonise chickens were each inoculated into groups of five birds. Three strains again did not colonise any of the chickens and the fourth strain colonised four out of the five chickens, but was poorly excreted. When infected chickens were placed in the same enclosure to facilitate interchange of strains, C. jejuni strain 331 was found to be dominant and colonised all 12 chickens by 21 days, displacing all other strains. C. jejuni strain 331, was then inoculated into groups of five birds with previously established colonisation by C. jejuni and C. coli strains. Strain 331 was able to replace the C. jejuni strain in all five birds but established co-colonisation with C. coli strain. Naturally occurring co-colonisation by two C. jejuni strains was detected in one chicken out of 200 tested. There was no obvious correlation between the type of DNA polymorphism in strains of chicken origin and their ability to colonise chickens.

Animals↗
PubMed1998

Survival and growth of Campylobacter jejuni after artificial inoculation onto chicken skin as a function of temperature and packaging conditions.

Campylobacter jejuni is one of the major causes of food poisoning in humans. C. jejuni is also widespread in food animals, and meat and meat products derived from food animals are the most common vector of bacterial transmission to humans. To determine the role of packing and storage conditions on the replication of C. jejuni on chicken, the virulent strain C. jejuni 81116 was artificially inoculated onto chicken skin pieces (1 cm2) and stored at different temperatures and under various packaging conditions. C. jejuni 81116 remained viable at -20 and -70 degrees C and was able to replicate at 4 degrees C and at ambient room temperature. C. jejuni 81116 was also inoculated onto chicken skin and subjected to repeated freeze thawing and the viability of the inoculum was quantified. C. jejuni 81116 could withstand repeated freeze thawing similar to that which may occur in the domestic home. Under all freezing conditions, C. jejuni 81116 retained a high level of viability and quickly replicated to levels which exceeded Australian food authorities' permitted bacteria level on raw food products after the sample was thawed.

Animals↗
PubMed1995

Antibacterial proteins from porcine polymorphonuclear neutrophils.

Antibacterial peptides were purified from porcine neutrophil granules collected from healthy pigs. Granule proteins, extracted with 0.2 mol/L sodium acetate were subjected to ion-exchange chromatography and five peaks (designated A to E) were detected. Individual porcine neutrophil granule proteins were shown to inhibit the growth of target organisms Escherichia coli and Staphylococcus aureus. The antimicrobial activity was shown to be concentration and time dependent. Peak D showed strong antimicrobial activity against S. aureus and peak C (with a greater number of eluted proteins) was shown to be active against both S. aureus and E. coli. One of the peptides was purified further by reverse-phase HPLC from peak fraction C. The MW of this peptide was approximately 5500 Da as determined by SDS-PAGE and mass spectral analysis and was active against both E. coli and S. aureus in vitro sustaining a > 90% decrease, respectively, in CFU after a 2 h exposure with 50 micrograms of this peptide. Amino acid analysis showed the peptide was rich in aspartate/aspartic acid, glutamine/glutamic acid, proline, arginine and threonine. The antimicrobial activity of this peptide and other novel proteins in porcine neutrophilic granules demonstrates the probable role of these proteins and peptides in host defence of porcine neutrophils against bacterial infection.

Amino Acids↗
PubMed1995

Characterization of immunorelated peptides to porcidin P1.

Porcidin P1, an antimicrobial peptide purified from the granules of porcine polymorphonuclear neutrophils (PMN) using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC), was covalently conjugated to BSA and used to generate monospecific polyclonal ascites. Antibodies raised against porcidin P1 were covalently coupled to an Affi-gel Hz affinity column and used for immunoaffinity chromatography of peptides from porcine PMN cell extract. Eleven immunorelated peptides were eluted from the column from neutrophil cell extracts and purified to homogeneity by HPLC. The molecular weights of the immunorelated peptides were determined by mass spectral analysis and ranged in size from 1.91 to 10.65 kDa. Of the 11 immunorelated peptides which were bound to the affinity column, only six peptides were recognized by the anti-porcidin antibodies after HPLC purification. Three immunoreactive peptides displayed potent antibacterial activity towards Staphylococcus aureus and Escherichia coli, reducing viability by as much as 99.9% (> 3 log reduction in CFU) when 5 mu g/mL of each purified peptide was used. The polyclonal monospecific antibodies also reacted with proteins from ovine and human PMN, illustrating possible structural relationships between small antibacterial peptides from the different species.

Amino Acids↗
PubMed1995

Campylobacter hyoilei sp. nov., associated with porcine proliferative enteritis.

Campylobacter hyoilei sp. nov. is the name proposed for an organism formerly described as strain RMIT 32AT (T = type strain) and a group of similar bacteria isolated from intestinal lesions of pigs with proliferative enteritis. The phenotypic characteristics of these organisms indicated that they are closely related to each other and are not strains of other Campylobacter spp. commonly isolated from pigs. The results of probing of ClaI-, EcoRV-, or BglII-cleaved genomic DNAs from C. hyoilei strains with a radiolabeled DNA probe that distinguishes between Campylobacter jejuni and Campylobacter coli indicated that C. hyoilei and C. coli are closely related. However, the 16S rRNA sequence of the reference strain of C. hyoilei, RMIT 32AT, was four bases different from the 16S rRNA sequence of C. jejuni CCUG 11284T and five bases different from the 16S rRNA sequence of C. jejuni subsp. doylei CCUG 24567T, suggesting that C. hyoilei is more closely related to C. jejuni than to C. coli. Hybridization between DNA from C. hyoilei type strain RMIT 32A and DNAs from selected type and reference strains of other Campylobacter species and subspecies, including C. jejuni, C. jejuni subsp. doylei, C. coli, Campylobacter mucosalis, and Campylobacter hyointestinalis, as well as the other C. hyoilei strains (the RMIT 32AT-like isolates), revealed that high levels of DNA hybridization (> 70%) occurred only between the reference strain and other strains of C. hyoilei.

Animals↗
PubMed1995

Differentiation of Campylobacter jejuni and Campylobacter coli strains by using restriction endonuclease DNA profiles and DNA fragment polymorphisms.

The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolates from poultry and humans were analyzed by using DNA restriction endonucleases ClaI and EcoRV. The DNA restriction patterns produced by ClaI and EcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains of C. jejuni tested. The DNA restriction patterns were further examined by Southern blot analysis with a previously constructed DNA probe, pMO2005, which is also able to distinguish between C. jejuni and C. coli spp. (5). Two types of patterns were produced by hybridization with the ClaI-cleaved DNA of C. jejuni strains, one of a single 18.5-kb genomic fragment and the other of 14.5- and 4.0-kb fragments. This indicated the presence of an extra ClaI site in this genomic fragment in the strains with the duplex pattern. The Southern blot analysis of 169 C. jejuni and C. coli isolates from poultry and from humans with DNA probe pMO2005 demonstrated that 78% of C. jejuni strains isolated from chickens hybridized with DNA probe pMO2005 with a characteristic 14.5- and 4.0-kb banding pattern and 22% hybridized with a single 18.5-kb fragment, whereas 71% of human isolates hybridized with the single 18.5-kb fragment and only 29% hybridized with 14.5- and 4.0-kb fragments. These findings suggest that only a small proportion of C. jejuni strains that colonize chickens may cause disease in humans.

Animals↗
PubMed1994

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.

Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae.

Animals↗
PubMed1993

Characterization of two DNA probes specific for Serpulina hyodysenteriae.

Two DNA probes, one 1.1- and one 0.75-kb probe, specific for Serpulina hyodysenteriae were isolated from a genomic library generated from virulent S. hyodysenteriae 5380. These probes are highly specific and react with all S. hyodysenteriae strains tested. Under stringent conditions, the DNA probes did not react with the nonpathogenic species Serpulina innocens or with other species of enteric bacteria, including Escherichia coli. Both probes are able to detect S. hyodysenteriae in colony blot hybridizations, and when applied to fecal specimens, they can detect 10(4) S. hyodysenteriae cells in 0.1 g of seeded fecal matter. Both probes can detect S. hyodysenteriae in fecal specimens from swine with clinical signs of swine dysentery after experimental challenge and from swine from a herd with an acute outbreak of swine dysentery. These probes have application as a diagnostic tool in veterinary microbiology.

Animals↗