B-Cell Epitope Mapping Using a Library of Overlapping Synthetic Peptides in an Enzyme-Linked Immunosorbent Assay.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20 mer sequence of the VapA protein.

Salmonella in the tropical household environment--Everyday, everywhere.

OBJECTIVES: To determine the prevalence of Salmonella in the environment of case and control houses, and compare serovars isolated from cases and their houses. METHODS: From 2005 to 2008, we tested samples from houses of 0-4 year old cases and community controls in Darwin and Palmerston for Salmonella. Case isolates were compared with environmental isolates. S. Ball and S. Urbana isolates were compared using Multiple Amplification of Phage Locus Typing (MAPLT) and Multiple-Locus Variable number of tandem repeat Analysis (MLVA). RESULTS: Salmonella were found in 47/65 (72%) case houses and 18/29 (62%) control houses; these proportions were not significantly different. In 21/47 (45%) houses, case and environmental isolates (from animal faeces, soil and vacuums) were indistinguishable. Multiple serovars were isolated from 20 (31%) case and 6 (21%) control houses. All but one environmental isolate are known human pathogens in the Northern Territory (NT). Each of the four pairs of S. Ball and S. Urbana were indistinguishable. CONCLUSIONS: Animal faeces were the most likely source of salmonellosis in cases. The similar prevalence of house isolates suggests that Salmonella is ubiquitous in this environment. The distinction of S. Ball and S. Urbana subtypes enabled linkage of human illness to environmental exposure. Environmental contamination with Salmonella is an important source of sporadic infection in children in the tropics.

The isolation and characterization of Campylobacter jejuni bacteriophages from free range and indoor poultry.

Six hundred and sixty one samples - primarily fresh chicken faeces - were processed to isolate wild type Campylobacter jejuni bacteriophages, via overlay agar methods using C. jejuni NCTC 12662. The aims of this study were to isolate and purify bacteriophages and then test for their ability to lyse field strains of C. jejuni in vitro. Of all samples processed, 130 were positive for bacteriophages. A distinct difference was observed between samples from different poultry enterprises. No bacteriophages could be isolated from indoor broilers. The majority of bacteriophages were isolated from free range poultry - both broilers and egg layers. Bacteriophages were purified and then selected for characterization based on their ability to produce clear lysis on plaque assay, as opposed to turbid plaques. Two hundred and forty one C. jejuni field isolates were tested for sensitivity to the bacteriophages. Lysis was graded subjectively and any minimal lysis was excluded. Using this system, 59.0% of the C. jejuni isolates showed significant sensitivity to at least one bacteriophage. The sensitivity to individual bacteriophages ranged from 10.0% to 32.5% of the C. jejuni isolates. Five bacteriophages were examined by electron microscopy and determined to belong to the Myoviridae family. The physical size, predicted genetic composition and genome size of the bacteriophages correlated well with other reported Campylobacter bacteriophages. The reasons for the observed difference between indoor broilers and free range poultry is unknown, but are postulated to be due to differences in the Campylobacter population in birds under different rearing conditions.

A new methodology for differentiation and typing of closely related Salmonella enterica serovar Heidelberg isolates.

This study describe the use of a combination of two recently proposed typing approaches, multiple amplification of prophage locus typing (MAPLT) and multiple-locus variable-number tandem-repeat analysis (MLVA) for subdividing within Salmonella enterica serovar Heidelberg (S. Heidelberg). The combined typing method was compared with pulsed-field gel electrophoresis (PFGE) by Simpson's index of diversity (DI). PFGE was shown to have a DI = 0.84 and was poor at differentiation of the predominant PT1 (Phage Type 1) phenotype. In comparison, the combined MAPLT/MLVA method comprising 3 MLVA and 9 MAPLT primer pairs provided a higher differentiating ability DI = 0.92. More importantly, the combined methodology was found to be superior in the differentiation of the predominant PT1 isolates. In conclusion, this study demonstrated the potential of the rapid and simple amalgamated MAPLT/MLVA approach in determining transmission of isolates of clonal phage type groups from various environmental sources to humans.

Antimicrobial and heavy metal resistance in commensal enterococci isolated from pigs.

Antibiotic resistance in animal isolates of enterococci is of public health concern because of the risk of transfer of antibiotic resistance isolates or resistance determinants to consumers via the food chain. In this study, phenotypic and genotypic resistance in 192 pig isolates of enterococci to ampicillin, avilamycin, avoparcin, bacitracin, flavophospholipol, gentamicin, narasin, tetracycline, tiamulin, tylosin, vancomycin, virginiamycin, copper and zinc were investigated by susceptibility test and molecular methods. Resistance rates varied between the species but all isolates were susceptible to ampicillin, avilamycin, avoparcin, gentamicin and narasin but resistant to tetracycline and tylosin and intermediately resistant to copper. Only Enterococcus gallinarum and Enterococcus casseliflavus were resistant to vancomycin and virginiamycin resistance was present in less than half the Enterococcus faecium isolates. Zinc resistance was largely confined to Enterococcus faecalis but bacitracin resistance was uncommon in E. faecalis in comparison with the other species. Tiamulin resistance was common in all species except E. casseliflavus. Resistance to flavophospholipol was detected in most E. faecium isolates and in a high proportion of E. gallinarum, E. casseliflavus and E. hirae/durans but was only found in one isolate of E. faecalis. No tetO, rplC, rplD, vanA, vanB, vatA and vatD genes were found. The presence of ermB, tetL, tetM, tcrB, aac6-aph2, tetK, tetS, vanC1, vanC2, lsaA, lsaB and vatE varied between the species and largely corresponded to the susceptibility phenotype. The findings show that resistance to antibiotics of high clinical significance for nosocomial Enterococcus infections is absent, whereas antimicrobial resistance was detected for some other antibiotics including bacitracin, flavophospholipol, tetracycline, tiamulin, tylosin and virginiamycin.

MLVA and phage typing as complementary tools in the epidemiological investigation of Salmonella enterica serovar Typhimurium clusters.

In South Australia serotyping and phage typing are employed for routine Salmonella surveillance. Molecular techniques such as Multiple-locus variable number tandem repeat analysis (MLVA) are increasingly utilized to aid outbreak investigations. During 2007 three Salmonella enterica serovar Typhimurium outbreaks involving phage types DT9, DT29, and DT44 were investigated. Human, food and environmental isolates were also typed by MLVA. In the DT9 outbreak cluster MLVA demonstrated distinct groupings that corresponded to epidemiological differences in time, place, and descriptive information on potential transmission mechanisms. In contrast, the human and food isolates of both the DT29 and DT44 clusters had identical MLVA profiles for all but one case. These data correlated with the epidemiology suggesting that these isolates were closely related and probably a single agent. These findings illustrate that phage typing and MLVA can provide different but complementary information for epidemiological investigations of Salmonella outbreaks.

Whole-genome sequencing and gene mapping of a newly isolated lytic enterococcal bacteriophage EFRM31.

Bacteriophages contribute greatly to bacterial evolution. There has been limited investigation of enterococcal bacteriophages, and only two enterococcal bacteriophages have been sequenced completely. In this study, a novel enterococcal bacteriophage, EFRM31, was isolated from a piggery effluent sample and then characterized. The complete bacteriophage genome was determined by shotgun sequencing. EFRM31 belongs to the family Siphoviridae (order Caudovirales) and has a circular double-stranded DNA genome. The putative EFRM31 genome consists of 16945 nucleotides with a low GC content (34.5%) and does not contain CpG islands. The EFRM31 genome contains 82 putative open reading frames, including 17 with identities to genes required for the assembly of a head-tail bacteriophage and 6 hypothetical proteins of unknown function. In general, the sequencing results from EFRM31 revealed considerable similarity to another enterococcal bacteriophage, EFAP-1. This identity and the order of shared genes suggest a close relationship or a common ancestor for these two bacteriophages.

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.

Linear B-cell epitope mapping using enzyme-linked immunosorbent assay for libraries of overlapping synthetic peptides.

The aim of this chapter is to provide a strategy for mapping linear antibody epitopes of protein antigens in order to discover candidates for vaccines or diagnostic tests. A set of overlapping peptides was designed and synthesised based upon a known amino acid sequence of the target protein, virulence-associated protein A (VapA) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals.The peptides were biotinylated and used in an ELISA to screen immune sera from foals. These biotinylated peptides were coated directly onto micro titre plates that had been pre-coated with NeutrAvidin. A linear B-cell epitope was identified by a universal recognition of sera to the synthetic peptides which corresponds to a particular fragment of the VapA protein.

A comparison of two PCR-based typing methods with pulsed-field gel electrophoresis in Salmonella enterica serovar Enteritidis.

Two novel molecular typing methods, multiple-locus variable-number tandem repeats (VNTR) analysis (MLVA) and multiple amplification of phage loci typing (MAPLT), were compared with pulsed-field gel electrophoresis (PFGE) for the discrimination of 128 Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates. Selected epidemiologically unrelated isolates represented a cross-section of phage types routinely isolated in Australia and included 28 isolates that could not be assigned a phage type. Targeting 5 previously described loci, MLVA generated 61 different profiles with a Simpson index of diversity of DI=0.968. MLVA locus STTR-5 proved to be the most diverse with 11 different alleles detected with a Nei's diversity index value of D=0.769. Using 8 MAPLT primers previously developed for S. Typhimurium produced 36 different profiles with a DI value of 0.948. By contrast, PFGE only generated 13 different pulsed-field patterns, DI=0.873. Within each phage type there was variation in the extent to which either molecular method was able to discriminate the S. Enteritidis isolates. MAPLT provided more discrimination in terms of the number of profiles and DI value for phage type 1 and the untypable strains while MLVA was more discriminatory for phage types 14var and 26. There was a general lack of concordance of either molecular assay to phage type. These results suggest that both MAPLT and MLVA have excellent potential as tools for epidemiological studies of S. Enteritidis.

The use of MAPLT and MLVA analyses of phenotypically closely related isolates of Salmonella enterica serovar Typhimurium.

The resolving power of multiple-locus variable-number tandem repeat analysis (MLVA) was undertaken on 78 phenotypically closely related isolates of Salmonella enterica serovar Typhimurium that were previously analysed with multiple amplification of phage locus typing (MAPLT) and pulsed-field gel electrophoresis (PFGE). Isolates were tested with 10 primer sets targeting tandem repeat loci in the S. Typhimurium genome. A new primer set targeting the SB21(ST64B) locus was also assessed for MAPLT analysis. Both methods produced similar levels of discrimination between 41 non-DT 126 isolates while MLVA proved superior in separating the DT 126 isolates. Results showed that two loci, STTR5 and STTR6, provided the most variation in tandem repeat numbers between the 78 S. Typhimurium isolates with 11 alleles detected for each locus. Some isolates did not produce amplified product with PCR for various loci, providing another level for discrimination. The remaining MLVA loci provided less allelic variation, with 4 loci failing to provide any discrimination. There were a number of isolates that were shown to be unique by one method but were clustered by the other method. Combining the most variable regions of both MAPLT and MLVA into one assay may provide significant resolution of isolates with the minimal number of primers utilised.

A comparison of three molecular typing methods for the discrimination of Salmonella enterica serovar Infantis.

Seventy-six epidemiologically unrelated Salmonella enterica serovar Infantis (S. Infantis) isolates were typed by pulsed-field gel electrophoresis (PFGE), multiple amplification of phage loci typing (MAPLT) and multiple-locus variable-number tandem-repeat analysis (MLVA). PFGE, using the restriction endonuclease XbaI, generated 23 different profiles for the 76 isolates (DI=0.848). MAPLT was undertaken using a combination of 11 primer sets based on bacteriophage sequences and generated 28 different profiles (DI=0.938). By contrast, MLVA only produced nine profiles (DI=0.668) with 13 different primer sets, including the five primer sets routinely used for S. Typhimurium typing. Reducing the number of MAPLT primer sets to four still provided a diversity index of 0.838. All three typing methods revealed two distinct lineages of S. Infantis, with most isolates demonstrating genetic traits of either lineage but not both. The results demonstrate that MAPLT can potentially provide greater discrimination and separation of S. Infantis isolates than both PFGE and MLVA. Furthermore, MAPLT data can be generated much more rapidly and with reduced labour input than PFGE and without the need for expensive PFGE electrophoresis equipment, nor does it require capillary sequencing of PCR fragments to accurately determine PCR fragment lengths as is the case with MLVA.

Chimeric vapA/groEL2 DNA vaccines enhance clearance of Rhodococcus equi in aerosol challenged C3H/He mice.

Rhodococcus equi remains a significant bacterial pathogen, causing severe pyogranulomatous pneumonia in foals aged 1-3 months. There is no effective vaccine currently available for the prevention of R. equi pneumonia. DNA vaccines are known to offer specific advantages over conventional vaccines. The aim of this study was to demonstrate efficacy of our recombinant DNA vaccine candidates, namely pcDNA3-Re1, pcDNA3-Re3 and pcDNA3-Re5 by combining a heat shock protein GroEL2 to a virulence-associated protein A (VapA) from R. equi to protect C3H/He mice against the R. equi infection. VapA was shown to be strongly recognised by sera from pneumonic foals. All vaccines elicited at least a doubling of the IgG2a/IgG1 ratio in comparison to the controls, indicating a bias to the Th1 response, which is postulated to be crucial for bacterial clearance and protective immunity against intracellular pathogens including R. equi. In addition, the immunised mice showed a significant reduction in R. equi in their lungs at 7 days after the aerosol challenge in comparison to PBS treated mice. However, examination of lung pathology 14 days after the challenge showed no gross differences in pathological changes between the unvaccinated and vaccinated animals. The lack of significant pathological changes suggests that the precise level of protection against R. equi pneumonia in the murine model of infection may not represent a true effectiveness of the potential vaccine candidates, indicating the mouse may not be the ideal non-equine model for vaccine studies and (or) the incomplete immunogenic antigen of vapA-based DNA vaccine constructs that mount an inadequate cell-mediated immune response against the R. equi infection.

Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar typhimurium by the multiple amplification of phage locus typing technique.

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including Salmonella. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related Salmonella species. The presence of genomic sequences derived from stable temperate phages in Salmonella offers an alternative for MLST of Salmonella. We have used MLST of prophage loci in Salmonella enterica serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.

Immune response to vaccines based upon the VapA protein of the horse pathogen, Rhodococcus equi, in a murine model.

Rhodococcus equi is a significant pathogen in foals predominantly causing a pyogranulomatous bronchopneumonia. Many vaccine candidates have been tested for the prevention of R. equi disease in foals. However, none of these have been developed for widespread commercial use. Previous studies have shown that a Th1 immune response is imperative for the protection of foals against R. equi disease. In this study a DNA and a protein vaccine based upon the well-characterised R. equi virulence-associated protein VapA were developed. The vaccines were tested in the BALB/c murine model and the results showed that both vaccine candidates elicited a Th1-type response in the host. Upon coadministration of an IL-12 expression plasmid with the DNA vaccine, an increase in the Th1 response was observed. However, when mice were challenged with 1.5 x 10(7) virulent R. equi ATCC 33701 none of the vaccinated mice showed protection apart from the mice immunised with live R. equi. These results indicate that despite their immunogenicity the VapA-based DNA and recombinant protein vaccines developed in this study were unable to prevent bacterial replication following a high-dose systemic challenge with virulent R. equi in the BALB/c model.

ST64B is a defective bacteriophage in Salmonella enterica serovar Typhimurium DT64 that encodes a functional immunity region capable of mediating phage-type conversion.

The Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) defective bacteriophage ST64B has a putative immunity (immC) region consisting of cI, cro and cII-like genes. Since ST64B is widespread in S. Typhimurium, studies were undertaken to determine whether this region might be functional and influence phage typing results. Cloning of ST64B immC-like genes and their subsequent expression in S. Typhimurium DTs showed that this region is able to mediate phage-type conversion in DTs 41 and 44. This confirms the functionality of the immC region and the patterns of lysis produced by phage typing are consistent with the predicted mechanism of action of the encoded protein products.

The immunogenicity of Rhodococcus equi GroEL2-based vaccines in a murine model.

Rhodococcus equi is a significant intracellular bacterial pathogen in foals. However, at present there is no commercially available vaccine for the prevention of R. equi-induced disease in these animals. Studies have shown that GroEL based vaccines can afford protection against some intracellular pathogens. In this study, the R. equi gene encoding the heat shock protein GroEL2 was cloned and sequenced, with a view to using it as a vaccine candidate. The promoter region of the gene contained two copies of controlling inverted repeat of chaperone expression (CIRCE) motifs, which are well-recognised transcriptional regulators of bacterial heat shock proteins. The R. equi GroEL2 was expressed in E. coli BL21 DE3 with a C-terminal His-tag and sequenced to confirm its identity. The R. equi purified His-tagged GroEL2 protein and a groEL2-based DNA vaccine were used in separate experiments to immunise BALB/c mice. The recombinant protein-based vaccine elicited a mixed Th1/Th2 response whereas the DNA vaccine was found to elicit a predominantly Th1 biased immune response. However, when vaccinated mice were challenged intravenously with 1.5 x 10(7) R. equi neither vaccine elicited enhanced bacterial clearance from the spleen or liver in this model. The reasons for this apparent lack of success are discussed.

A fluorescent amplified fragment length polymorphism study of Salmonella enterica serovar Sofia, the major Salmonella serovar isolated from chickens in Australia.

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 68 isolates of Salmonella enterica subsp. salamae serovar Sofia (S. Sofia). Fifty eight isolates were obtained over a period of approximately 15 years from a range of human, chicken industry and environmental sources throughout Australia. A further ten isolates were identified from human and poultry sources in Israel from 1972 to 1987. Analysis of FAFLP profiles for fragments between 50 to 500 base pairs in length indicated distinct clusters of isolates. All but seven isolates clustered into four groups of >90% similarity and all isolates displayed at least 70% similarity with each other. No cluster could be attributed to a particular geographical, temporal or source-of-isolation origin. It is concluded that S. Sofia is genetically variable with certain clones persisting over time but no group appears unique to Australia.

Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64.

The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.

Development of a diagnostic PCR assay that targets a heat-shock protein gene (groES) for detection of Pseudomonas spp. in cystic fibrosis patients.

Laboratory detection of Pseudomonas spp., in particular Pseudomonas aeruginosa, remains an important assay in the management of patients with cystic fibrosis (CF). As the groES and groEL genes of P. aeruginosa have now been cloned and their nucleotide sequences determined, the aim of this study was to develop a novel PCR assay for the detection of Pseudomonas spp. from patients with CF by employing conserved primer regions of the groE heat-shock protein domain gene. A PCR assay was designed that targeted a 536 bp region of the groE gene to detect Pseudomonas spp. PCR amplification of genomic DNA from extracted organisms generated an amplicon of the expected size (approx. 536 bp) for all P. aeruginosa (n = 60), Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas stutzeri isolates examined, but did not produce a positive amplicon for several other genera and species that are commonly isolated from the sputum of CF patients. RFLP analysis of the amplicons of all P. aeruginosa isolates demonstrated a single RFLP type that consisted of three bands at approximately 80, 190 and 250 bp; direct sequencing of the amplicons demonstrated the presence of a single sequence type, indicating the highly conserved nature of this region. In addition, the assay successfully produced a positive signal from primary non-selective plates of three known P. aeruginosa culture-positive CF patients, but was unable to generate a signal in a further six CF patients who had no history of infection with P. aeruginosa or other Pseudomonas spp. This assay is recommended to detect the presence of Pseudomonas spp., including P. aeruginosa, from primary culture plates that originate from laboratory analysis of CF patients' sputum, particularly at review, in those patients with no previous history of Pseudomonas infection or those who appear to be transiently colonized by this organism. Employment of such molecular methodologies, in conjunction with routine clinical sputum cultures, may provide improved information on the microbial status of CF patients, which will aid clinicians in both optimum patient management in terms of antibiotic regimes and CF centre infection-control practices.