Molecular identification and characterisation of catalase and catalase-like protein genes in urease-positive thermophilic Campylobacter (UPTC).

BACKGROUND: Thermophilic Campylobacter are important bacterial pathogens of foodborne diseases worldwide. These organisms' physiology requires a microaerophilic atmosphere. To date, little is known about the protective catalase mechanism in urease-positive thermophilic campylobacters (UPTC); hence, it was the aim of this study to identify and characterise catalase and catalase-like protein genes in these organisms. MATERIALS AND METHODS: Catalase (katA) and catalase (Kat)-like protein genes from the Japanese UPTC CF89-12 strain were molecularly analysed and compared with C. lari RM2100 and other C. lari and thermophilic Campylobacter reference isolates. RESULTS: A possible open reading frame of 1,422 base pairs, predicted to encode a peptide of 474 amino acid residues, with calculated molecular weight of 52.7 kilo Daltons for katA, was identified within UPTC CF89-12. A probable ribosome binding site, two putative promoters and a putative ρ-independent transcription terminator were also identified within katA. A similar katA cluster also existed in the C. lari RM2100 strain, except that this strain carries no DcuB genes. However, the Kat-like protein gene or any other homologue(s) were never identified in the C. lari RM2100 strain, or in C. jejuni and C. upsaliensis. CONCLUSIONS: This study demonstrates the presence of catalase/catalase-like protein genes in UPTC organisms. These findings are significant in that they suggest that UPTC organisms have the protective genetic capability of helping protect the organisms from toxic oxygen stress, which may help them to survive in physiologically harsh environments, both within human and animal hosts, as well as in the natural environment.

Biochemical characterization of mitochondria from adult worms and plerocercoid larvae of Spirometra mansoni shows mixed populations of anaerobic and aerobic mitochondria.

The mitochondria of adult and plerocercoid Spirometra mansoni were characterized in isolated mitochondria and in situ by electron microscopic histochemistry with special attention to the respiratory chain. Although the specific activities of the constituent enzyme complexes of succinate oxidase are fairly similar in adult and plerocercoid mitochondria, those of succinate oxidase and NADH-FRD are approximately 4- and 25-fold higher in adult mitochondria than in plerocercoid mitochondria, respectively. Quinone analysis by high performance liquid chromatography and mass spectrometry showed that adult and plerocercoid mitochondria contained both rhodoquinone-10 and ubiquinone-10 at concentrations of 4.98 and 0.106 nmol mg-1 for adult, and 0.677 and 0.137 nmol mg-1 for plerocercoid, respectively. Inhibition studies on the succinate-oxidase system of adult mitochondria showed that they possessed both cyanide-sensitive and -insensitive succinate oxidases, the latter of which produces hydrogen peroxide. Adult mitochondria, when NADH was used as a substrate, were shown to produce hydrogen peroxide, and the production of hydrogen peroxide decreased to undetectable levels in the presence of fumarate. The specific activities of NADH-fumarate reductase and cytochrome c oxidase were significantly higher in mature proglottids than in immature and gravid proglottids. Isopycnic density-gradient centrifugation analyses and in situ electron microscopic histochemistry revealed that both adult and plerocercoid mitochondria were heterogeneous in terms of respiratory function and physicochemical properties. The physiological significance of adult and plerocercoid mitochondria is discussed in relation to the oxygen tension of their parasitic habitats.

Bacterial stress response to environmental radiation relating to the Fukushima radiation discharge event, Japan: will environmental bacteria alter their antibiotic susceptibility profile?

Antibiotic resistance in clinical pathogens in humans may be traced back to resistance mechanisms in environmental bacteria and any factors, which are likely to alter (upregulate) resistance in environmental organisms, is of potential and eventual consequence to human pathogens. Furthermore, sublethal doses of gamma radiation to environmental organisms may cause sublethal stress and a selective pressure, which may lead to mutational events that alter the bacterium's susceptibility profile. A gamma (γ) radiation simulation experiment was performed to emulate the exposure of four environmental bacteria, including Listeria innocua, Bacillus subtilis, E. coli and Pseudomonas aeruginosa, to levels of radiation in and around Fukushima, Japan, equating to 1, 10 and 100 years equivalence exposure. Alteration to susceptibility to 14 antibiotics was measured as the primary endpoint. There was no significant alteration in the susceptibility of the Gram-positive organisms, whereas both Gram-negative organisms became slightly more susceptible to the antibiotics tested over time. These data indicate that such radiation exposure will not increase the antibiotic resistance profile of these organisms and hence not add to the global public health burden of increased antibiotic resistance in human bacterial pathogens.

UVc-irradiation sublethal stress does not alter antibiotic susceptibility of staphylococci (MRSA, MSSA, and coagulase-negative staphylococci) to ß-lactam, macrolide, and fluoroquinolone antibiotic agents.

Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely ß-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.

Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis.

OBJECTIVES: Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). METHODS: The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. RESULTS: Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). CONCLUSIONS: Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.

Development of a new molecular detection method for Taylorella equigenitalis.

On PCR amplification of the intervening sequences (IVSs) in the central (helix 45) region within 23S rRNA gene sequences with T. equigenitalis (n = 34), as well as T. asinigenitalis (n = 35) and Bordetella (n = 11) isolates by using the primer pair of f-/r-23STis2, approximately 0.8 kb of the amplicons were generated, sequenced and analyzed. One IVS of approximately 70 bp in length was identified in all the Taylorella organisms but not Bordetella. PCR amplification was further developed for the convenient and rapid molecular detection of T. equigenitalis organisms with the IVS in the helix 45 region within the 23S rRNA genes as target by using the primer pairs (f-IVSde/r-23de). Thus, these results clearly demonstrated that PCR amplification with the primer pair (f-IVSde/r-23de) can be reliable in order to differentiate the T. equigenitalis isolates from both the T. asinigenitalis and Bordetella organisms.

A phylogenetic comparison of urease-positive thermophilic Campylobacter (UPTC) and urease-negative (UN) C. lari.

In the present study, the reliability of full-length gene sequence information for several genes including 16S rRNA was examined, for the discrimination of the two representative Campylobacter lari taxa, namely urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC). As previously described, 16S rRNA gene sequence are not reliable for the molecular discrimination of UN C. lari from UPTC organisms employing both the unweighted pair group method using arithmetic means analysis (UPGMA) and neighbor joining (NJ) methods. In addition, three composite full-length gene sequences (ciaB, flaC and vacJ) out of seven gene loci examined were reliable for discrimination employing dendrograms constructed by the UPGMA method. In addition, all the dendrograms of the NJ phylogenetic trees constructed based on the nine gene information were not reliable for the discrimination. Three composite full-length gene sequences (ciaB, flaC and vacJ) were reliable for the molecular discrimination between UN C. lari and UPTC organisms employing the UPGMA method, as well as among four thermophilic Campylobacter species.

15th International Workshop on Campylobacter, Helicobacter and Related Organisms.

The purpose of this communication is to update the veterinary public health community as to what poultry-related interventions were presented at the recent biennial International Workshop on Campylobacter, Helicobacter and Related Organisms (CHRO), which was held in Niigata, Japan, September 2-5, 2009. More than 30 years have passed since the publication of Martin Skirrow's seminal paper in the British Medical Journal in which he described Campylobacter enteritis as a new disease (1). This publication precipitated a global interest in thermophilic campylobacters. Three decades later, these organisms still pose a grave threat to public health. Furthermore, 10 years have passed since Parkhill et al. published the genome sequence of Campylobacter jejuni NCTC11168 (2).

Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.

Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals.

Molecular characterization of macrolide resistance determinants [erm(B) and mef(A)] in Streptococcus pneumoniae and viridans group streptococci (VGS) isolated from adult patients with cystic fibrosis (CF).

OBJECTIVES: Although long-term use of azithromycin has shown a significant clinical improvement for patients with cystic fibrosis (CF), its long-term effect on the susceptibility of commensal flora within CF airways has not yet been examined. We therefore suggest that long-term use of azithromycin increases macrolide resistance in commensal streptococci. METHODS: Erythromycin susceptibility in naturally colonizing viridans group streptococci (VGS) was characterized, as well as macrolide resistance gene determinants through sequence analysis, in pneumococci (n = 15) and VGS [n = 84; i.e. Streptococcus salivarius (n = 30), Streptococcus mitis (n = 17), Streptococcus sanguinis (n = 11), Streptococcus oralis (n = 10), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 3), Streptococcus infantis (n = 3), Streptococcus cristatus (n = 2), Streptococcus anginosus (n = 1) and Streptococcus australis (n = 1)] isolated from sputum from 24 adult CF patients, who were on oral azithromycin therapy for at least the previous 7 months. RESULTS: Almost three-quarters of isolates (74; 74.7%) were resistant to erythromycin, whilst a further 15 (15.2%) had reduced susceptibility, leaving only 10 (10.1%) isolates susceptible to erythromycin. The majority (89.8%) were not susceptible to erythromycin, as demonstrated by possession of the erm(B) gene in 25/99 (25.3%), the mef(A) gene in 1/99 (1.0%), the mef(E) gene in 75/99 (75.8%) and both erm(B) and mef(E) genes simultaneously in 11/99 (11.1%). These results indicate that genotypic resistance for macrolides is common in VGS in adult CF patients, with efflux being over three times more frequent. CONCLUSIONS: Long-term treatment with azithromycin in CF patients may reduce antibiotic susceptibility in commensal VGS, where these organisms may potentially act as a reservoir of macrolide resistance determinants for newly acquired and antibiotic-susceptible pathogens.

Identification and characterization of intervening sequences within 23S rRNA genes from more than 200 Campylobacter isolates from seven species including atypical campylobacters.

BACKGROUND: Identification and characterization of intervening sequences (IVSs) within 23S rRNA genes from Campylobacter organisms including atypical campylobacters were carried out using two PCR primer pairs, designed to generate helix 25 and 45 regions. RESULTS: Only C. sputorum biovar sputorum LMG7975 and fecalis LMG8531, LMG8534 and LMG6728 of a total of 204 Campylobacter isolates (n = 56 C. jejuni; n = 11 C. coli; n = 33 C. fetus; n = 43 C. upsaliensis; n = 30 C. hyointestinalis; n = 4 C. sputorum biovar sputorum; n = 5 C. sputorum biovar fecalis; n = 5 C. sputorum biovar paraureolyticus; n = 10 C. concisus; n = 7 C. curvus) were shown to carry IVSs in helix 25 region. C. sputorum biovar fecalis LMG8531 and LMG8534, interestingly, carried two different kinds of the 23S rRNA genes with and without the IVS, respectively. Consequently, in a total of 265 isolates of 269, including 65 C. lari isolates examined previously, the absence of IVSs was identified in the helix 25 region. In the helix 45 region, all the C. hyointestinalis, C. sputorum and C. concisus isolates were shown not to carry any IVSs. However, the 30 of 56 C. jejuni isolates (54%), 5 of 11 C. coli (45%), 25 of 33 C. fetus (76%), 30 of 43 C. upsaliensis (70%) and 6 of 7 C. curvus (90%) were shown to carry IVSs. In C. jejuni and C. upsaliensis isolates, two different kinds of the 23S rRNA genes were also identified to occur with and without IVSs in the helix 45 region, respectively. CONCLUSIONS: Secondary structure models were also constructed with all the IVSs identified in the present study. In the purified RNA fractions from the isolates which carried the 16S or 23S rRNA genes with the IVSs, no 16S or 23S rRNA was evident, respectively.

Structural analysis of the full-length gene encoding a fibronectin-binding-like protein (CadF) and its adjacent genetic loci within Campylobacter lari.

BACKGROUND: The combined sequences encoding a partial and putative rpsI open reading frame (ORF), non-coding (NC) region, a putative ORF for the Campylobacter adhesin to fibronectin-like protein (cadF), a putative Cla_0387 ORF, NC region and a partial and putative Cla_0388 ORF, were identified in 16 Campylobacter lari isolates, using two novel degenerate primer pairs. Probable consensus sequence at the -35 and -10 regions were identified in all C. lari isolates, as a promoter. RESULTS: Thus, cadF (-like) gene is highly conserved among C. lari organisms. Transcription of the cadF (-like) gene in C. lari cells in vivo was also confirmed and the transcription initiation site was determined. A peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins was completely conserved amongst the putative cadF (-like) ORFs from the C. lari isolates. CONCLUSION: The putative cadF (-like) ORFs from all C. lari isolates were nine amino acid larger than those from C. jejuni, and showed amino acid residues 137 -140 of FALG (50% identity), instead of the FRLS residues of the maximal fibronectin-binding activity site demonstrated within C. jejuni CadF. A neighbor joining tree constructed based on cadF (-like) gene sequence information formed a major cluster consisting of C. lari isolates, separating from the other three thermophilic campylobacters.

Demonstration of the absence of intervening sequences within 23S rRNA genes from Campylobacter lari.

Cloning, sequencing and characterization of nearly full-length 23S rRNA genes in 12 urease-positive thermophilic Campylobacter (UPTC) isolates were carried out using two novel PCR primer pairs. Nucleotide sequences of the 23S rRNA genes from the 12 isolates were first shown not to carry any intervening sequences (IVSs) in both the 25 and 45 helix regions. Then, two PCR primer sets were designed in silico for amplification of the helix 25 and 45 regions within 23S rRNA gene sequences from Campylobacter lari. No IVSs were identified within the 23S rRNA genes among a total of 53 isolates of C. lari, following PCR amplification, TA cloning and sequencing procedures. Intact 23S rRNA was identified in all 65 C. lari isolates, resulting in no production of the fragmented 23S rRNA. These data suggest that C. lari may not have any opportunity to interact with any other source of IVSs until now, or has been unable to integrate IVSs into their own genomes.

Cloning, sequencing and expression of full-length Campylobacter invasion antigen B gene operon from Campylobacter lari.

A novel PCR primer pair for amplification of full-length cia B gene from thermophilic campylobacters, generated an amplicon of approximately 2.2 kilo base pairs (kbp) with all 18 isolates (n = 7 for urease-negative (UN) C. lari; n = 9 urease-positive thermophilic Campylobacter (UPTC); n = 1 C. jejuni; n = 1 C. coli). The putative open reading frame (ORF) of the cia B from C. lari isolates consisted of 1,833 bp similarly, but differing from those of C. jejuni and C. coli isolates. The putative promoter structures consisting of a semi-conserved T -rich sequence and a consensus sequence at the -10 region were identified upstream of the putative ORF in all the C. lari isolates. A start codon ATG and a probable ribosome binding site were also identified in all the isolates. In addition, two distinctly different and taxon (UN C. lari and UPTC) dependent hypothetically intrinsic rho -independent transcriptional terminators for the cia B were identified to occur within the C. lari. Reverse transcription-PCR analysis identified the transcription of cia B gene in the C. lari cells. The neighbor joining tree suggested that the nucleotide sequence information of the cia B had molecular discrimination efficacy among UN C. lari, UPTC, C. jejuni and C. coli organisms.

Zoonoses associated with petting farms and open zoos.

The popularity of open farms and petting zoos has increased markedly over the last 5 years, with most children in developed countries now having the opportunity to visit such a facility at some stage in their childhood, either through school or family visits. The open access policy of these establishments allows visitors to be in direct contact with animals such as sheep (lambs), goats, cats (kittens), dogs (puppies), and birds and to have the opportunity to feed such animals. This contact may lead to the transmission of microbial pathogens from animals to humans, e.g., Escherichia coli O157:H7, resulting in human disease. This review outlines the causal organisms associated with such zoonoses, a description of previous outbreaks at farms and zoos, as well as infection control measures to help prevent such zoonotic infections.

Bactericidal activity of denture-cleaning formulations against planktonic health care-associated and community-associated methicillin-resistant Staphylococcus aureus.

The aim of this study was to examine the survival dynamics of several epidemic health care-associated (HA) and community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) in planktonic state in widely employed denture-cleaning solutions. The bacteriocidal activity of five widely employed denture-cleaning formulations were examined against five phage-types of HA-MRSA (EMRSA 15, EMRSA 16, Irish 1, Irish 2, unique type), as well as a CA-MRSA strain, in this study. Viable MRSA cells (circa 10(5) cfu/mL) were coincubated with optimum recommended working concentrations of denture-cleaning solutions for up to 17 hours (overnight). Recovery experiments were unable to isolate any of the inoculated MRSA organisms 10 minutes post inoculation. The significance and impact of this short study indicates that HA-MRSA and CA-MRSA are not able to remain culturable for 10 minutes in planktonic form, in commonly used denture-cleaning formulations widely available on the UK High Street, suggesting that these formulations may be useful in lowering the numbers of MRSA. Further work is however required to examine the more complex survival dynamics of MRSA in naturally derived denture biofilm, associated with dental plaque and the use of such cleaning formulations.

The epidemiology of antibiotic resistance in Campylobacter.

Antibiotic resistance, particularly with the fluoroquinolones and macrolide antibiotics, has now emerged globally with thermophilic campylobacters, including Campylobacter jejuni and C. coli, giving rise to concerns about how these organisms have acquired such resistance characteristics, as well as consequences for human and animal treatment. This review examines (i) the clinical epidemiology of antibiotic resistance in human and animal thermophilic campylobacters, (ii) an update on resistance rates globally, (iii) surveillance of antimicrobial resistance in campylobacters originating from animals, particularly poultry, (iv) the role of the environment in the acquisition and transmission of antibiotic-resistant campylobacters, as well as (v) issues of biocide resistance in campylobacters.

Genetic heterogeneity of urease gene loci in urease-positive thermophilic Campylobacter (UPTC).

Degenerate PCR primers in silico based on the two urease structural genes, ureA and ureB, were designed for urease-positive thermophilic Campylobacter (UPTC). Resultant PCR amplification employing these primers generated an amplicon of approximately 2kb, which was cloned and sequenced in UPTC (n=12) isolated from various parts of Europe and Japan. Overall, sequence similarities were shown to be 96.7 to 99.9%. Following sequence alignment analysis, the approximate 1.96kb regions were deduced to consist of parts of ureA (about 570bps) and ureB (about 1390bps) with an overlapping region between the ureA and ureB gene loci. Although a total of 144 heterogeneous sites of all substitutions were located throughout this region, the substitution ratio was higher in the ureA region (1/Omega10bases) than in the ureB region (1/Omega15bases). A resulting dendrogram was constructed, which was based on the nucleotide sequence data of 12 UPTC isolates and demonstrated that the UPTC were genetically variable. They formed a major cluster with Helicobacter, separate from the other urease-producing bacteria examined, suggesting a shared ancestry between UPTC and Helicobacter.

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses.

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.

Molecular analysis of the tlyA gene in Campylobacter lari.

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.