The effect of periodontal scaling and root polishing on serum IL-17E concentrations and the IL-17A:IL-17E ratio.

OBJECTIVES: The serum IL-17A:IL-17E ratio has previously been demonstrated to be a clinical marker of periodontitis. The aim of this study was to determine the effects of non-surgical periodontal treatment on the serum IL-17A:IL-17E ratio. MATERIALS AND METHODS: Forty chronic periodontitis patients completed this study and received periodontal treatment comprising scaling and root planing plus ultrasonic debridement. Clinical data were recorded at baseline, 6 weeks (R1) after treatment completion (full-mouth or quadrant-scaling and root planing) and 25 weeks after baseline (R2). Serum samples were taken at each time point and cytokines concentrations determined by ELISA. RESULTS: Following treatment, statistically significant reductions were noted in clinical parameters. However, IL-17A and IL-17E concentrations were significantly greater than baseline values before- and after-adjusting for smoking. The IL-17A:IL-17E ratio was lower at R1 and R2. Serum IL-6 and TNF levels were significantly lower at R1 only. Also exclusively at R1, serum IL-17A and IL-17E correlated positively with clinical parameters, while the IL-17A:IL-17E ratio correlated negatively with probing pocket depth and clinical attachment. CONCLUSION: Increased serum IL-17E and a reduced IL-17A:IL-17E ratio may be indicative and/or a consequence of periodontal therapy. Therefore, the role of IL-17E in periodontal disease progression and the healing process is worthy of further investigation. CLINICAL RELEVANCE: IL-17E may be a valuable biomarker to monitor the healing process following periodontal treatment as increased IL-17E levels and a reduced IL-17A:IL-17E ratio could reflect clinical improvements post-therapy. Therefore, monitoring serum IL-17E might be useful to identify individuals who require additional periodontal treatment.

Risk factors for bovine periodontal disease - a preliminary study.

The work presented in this pilot study aimed to identify potential risk factors associated with bovine periodontitis development. Bovine periodontitis is a multifactorial polymicrobial infectious disease for which the aetiopathogenesis and risk factors are not fully understood. From cattle slaughtered in an abattoir in Scotland, 35 dental arcades with periodontal lesions and 40 periodontally healthy arcades were selected over seven visits for study. Multivariable logistic regression analysis was used to evaluate the association between periodontitis and the independent variables, gender, age and breed. For every increase in year of age, cattle were 1.5 times more likely to have periodontitis. A graphical analysis indicated that within the limits of this study, we could not detect any major influence of breed on the age-effect. Although logistic regression analysis demonstrated that periodontitis lesions are more prevalent with increasing age of cattle the underlying mechanisms remain unclear. It is likely that periodontitis is an important cause of oral pain in older cattle and can contribute to reduced productivity/performance. Further studies with a larger sample size are necessary to elucidate the associations between potential risk factors and periodontitis in cattle and to define its effects on animal welfare and productivity.

Molecular detection of transcriptionally active bacteria from failed prosthetic hip joints removed during revision arthroplasty.

The purpose of this study was to use microbiological culture and bacterial 16S rRNA gene sequencing methods to detect transcriptionally active bacteria present on the surface of failed prosthetic hip joints removed during revision arthroplasty. Five failed prosthetic hip joints were sonicated to dislodge adherent bacteria and subjected to microbiological culture. Bacterial RNA was extracted from each sonicate, cDNA prepared by reverse transcription and the 16S rRNA gene amplified using universal primers. Polymerase chain reaction (PCR) products were cloned, assigned to distinct groups by restriction fragment length polymorphism (RFLP) analysis and one representative clone from each group was sequenced. Bacteria were identified by comparison of the obtained 16S rRNA gene sequences with those deposited in public access sequence databases. All five specimens were positive for the presence of bacteria by both culture and PCR. Culture methods identified species from eight genera. Molecular detection of transcriptionally active bacteria identified a wider range of species. A total of 42 phylotypes were identified, of which Lysobacter gummosus was the most abundant (31.6%). Thirty-four clones (14.5%) represented uncultivable phylotypes. No potentially novel species were identified. It is concluded that a diverse range of transcriptionally active bacterial species are present within biofilms on the surface of failed prosthetic hip joints.

Molecular identification of bacteria on the tongue dorsum of subjects with and without halitosis.

AIM: Compare the microbial profiles on the tongue dorsum in patients with halitosis and control subjects in a UK population using culture-independent techniques. MATERIALS AND METHODS: Halitosis patients were screened according to our recently developed recruitment protocol. Scrapings from the tongue dorsum were obtained for 12 control subjects and 20 halitosis patients. Bacteria were identified by PCR amplification, cloning and sequencing of 16S rRNA genes. RESULTS: The predominant species found in the control samples were Lysobacter-type species, Streptococcus salivarius, Veillonella dispar, unidentified oral bacterium, Actinomyces odontolyticus, Atopobium parvulum and Veillonella atypica. In the halitosis samples, Lysobacter-type species, S. salivarius, Prevotella melaninogenica, unidentified oral bacterium, Prevotella veroralis and Prevotella pallens were the most commonly found species. For the control samples, 13-16 (4.7-5.8%) of 276 clones represented uncultured species, whereas in the halitosis samples, this proportion increased to 6.5-9.6% (36-53 of 553 clones). In the control samples, 22 (8.0%) of 276 clones represented potentially novel phylotypes, and in the halitosis samples, this figure was 39 (7.1%) of 553 clones. CONCLUSIONS: The microflora associated with the tongue dorsum is complex in both the control and halitosis groups, but several key species predominate in both groups.

Gingival Toll-like receptor and cytokine messenger RNA levels in equine periodontitis and oral health.

REASONS FOR PERFORMING STUDY: Equine periodontitis is a common and painful condition. However, the disease often goes unnoticed by owners and is thus a major welfare concern. The aetiopathogenesis of the condition remains poorly understood and has been investigated in few studies. The innate immune system is known to play an important role in human periodontitis, but its role in equine periodontitis has not been examined. OBJECTIVES: To quantify the messenger (m)RNA levels of Toll-like receptors (TLRs) and cytokines in gingival tissue from orally healthy horses and those affected by periodontitis. STUDY DESIGN: Observational study. METHODS: Gingival tissue samples were taken post-mortem from 13 horses with no clinical signs of oral disease and 20 horses with periodontitis. mRNA levels of TLR2, TLR4 and TLR9 and cytokines interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), IL-4, IL-6, IL-10, IL-12, IL-17 and interferon-γ (IFN-γ) were determined using quantitative real-time PCR. The statistical significance of results was assessed using appropriate t tests. RESULTS: mRNA levels of all TLRs and cytokines were upregulated in equine periodontitis. Significant increases in mRNA levels of TLR2, TLR9, IL-4, IL-10, IL-12 (P≤0.05) and IFN-γ (P≤0.01) were observed for both unweighted and age-weighted analyses of diseased gingival tissue samples compared with healthy gingival samples. In comparisons of samples of periodontitis lesions with healthy gingival control samples from the same horse, significant increases in mRNA levels of TLR4, TLR9, IL-10, IFN-γ (P≤0.05), TLR2, IL-1ß and IL-12p35 (P≤0.01) were observed. CONCLUSIONS: This study has provided an initial insight into the involvement of the immune system in equine periodontitis. Increased mRNA levels of TLR2, TLR4 and TLR9 indicate substantial microbial challenge in diseased gingival tissue. A mixed Th1/Th2/Th17 cytokine response is produced in equine periodontitis. Further studies are required to more fully characterise the role of the innate immune system in this disease.

A novel species-specific PCR assay for identifying Lactobacillus fermentum.

Lactobacillus fermentum is a Gram-positive bacterium that is associated with active caries lesions. Methods for identifying Lactobacillus species traditionally have been based upon culture methods coupled with biochemical tests, which are generally unreliable. The aim of this study was to develop a species-specific PCR assay for the direct detection of L. fermentum in oral clinical samples. PCR primers specific for L. fermentum were identified by alignment of bacterial 16S rRNA genes and selection of sequences specific for L. fermentum at their 3' ends. PCR positivity for L. fermentum DNA was indicated by amplification of a 337 bp product. The primers were shown to be specific for L. fermentum DNA, since no PCR product was obtained when genomic DNA from a wide range of other oral bacteria, including closely related Lactobacillus species, were used as test species. The PCR assay was then used in an attempt to identify L. fermentum DNA in supragingival plaque samples and in pus aspirates from subjects with acute dento-alveolar abscesses. Four out of 70 (5.7 %) supragingival plaque samples analysed were positive for the presence of L. fermentum DNA while none of the 19 pus samples analysed was positive for L. fermentum DNA. This PCR assay provides a more rapid, specific and sensitive alternative to conventional culture methods for the identification of L. fermentum in clinical specimens.

Microbiological culture analysis of the tongue anaerobic microflora in subjects with and without halitosis.

OBJECTIVE: Determination of the microflora present on the tongue dorsum of subjects with and without halitosis using conventional microbiological culture methods. METHODS: Twenty-one halitosis and 20 control patients were recruited using a strict clinical protocol. Samples were collected from the posterior dorsum of the tongue using a sterile brush. Each sample was vortex mixed for 30 s and serial 10-fold dilutions to 10(-7) were carried out. Samples were plated onto fastidious anaerobe agar (FAA) and FAA enriched with vancomycin. These were incubated under anaerobic conditions for 10 days at 37 degrees C. Strict anaerobes were identified by metronidazole sensitivity and bacteria were identified to genus level by a combination of colony morphology, Gram staining and biochemical and enzymatic tests (rapid ID 32 A). RESULTS: The predominant species in test and control groups were Veillonella sp. and Prevotella sp. Greater species diversity was found in the halitosis samples compared with controls. The halitosis samples contained an increased incidence of unidentifiable Gram-negative rods, Gram-positive rods and Gram-negative coccobacilli. CONCLUSIONS: There was no obvious association between halitosis and any specific bacterial genus. The increased species diversity found in halitosis samples suggests that halitosis may be the result of complex interactions between several bacterial species. The role of uncultivable bacteria may also be important in contributing to this process.

Sequences of the ribonucleotide reductase-encoding genes of equine herpesvirus 4.

The equine herpesvirus 4 (EHV-4) genes encoding the two subunits of the enzyme ribonucleotide reductase (RR) were cloned and their nucleotide (nt) sequences determined. The large subunit (RR1) is predicted to comprise 789 amino acids (aa), which compares with lengths of 790, 775 and 1137 aa for the RR1 proteins encoded by equine herpesvirus 1 (EHV-1) gene 21, varicella zoster virus (VZV) gene 19 and herpes simplex virus type 1 (HSV-1) UL39, respectively. In common with VZV RR1, the EHV-4 RR1 protein lacks the N-terminal domain of HSV-1 RR1 which possesses protein kinase activity. EHV-4 RR1 demonstrates identities of 88, 52 and 29% with the RR1 proteins of EHV-1, VZV and HSV-1, respectively. The small subunit (RR2) is predicted to be 320 aa in length, which compares with lengths of 321, 306 and 340 aa for the RR2 proteins encoded by EHV-1 gene 20, VZV gene 18 and HSV-1 UL40, respectively. The EHV-4 RR2 protein exhibits identities of 90, 60 and 55% with the RR2 proteins of EHV-1, VZV and HSV-1, respectively.

Development of a PCR assay specific for Peptostreptococcus anaerobius.

Peptostreptococcus anaerobius is a gram-positive anaerobic coccus that is widely distributed in the normal human flora. The organism has also been implicated as a causative agent of several systemic infections, including endocarditis and infections of the genitourinary and gastrointestinal tracts. Its role in oral disease is less well defined, although it has been implicated in periodontal disease, gingivitis and root canal infections. Identification of P. anaerobius in clinical samples is currently reliant upon traditional culture and biochemical methods. The aim of this study was to develop a novel PCR assay for the detection of P. anaerobius and to attempt detection of this organism in oral samples. PCR primers specific for P. anaerobius DNA were developed by alignment of bacterial 16S ribosomal RNA gene sequences and selection of sequences specific at their 3' ends for P. anaerobius. When used in a PCR assay, positivity for P. anaerobius DNA was indicated by the amplification of a 943-bp product. The primers were shown to be specific for P. anaerobius DNA, as no PCR products were obtained when genomic DNA from a wide range of other Peptostreptococcus species and other oral bacteria were used as templates. The PCR assay was then applied to the detection of P. anaerobius DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. All of 60 subgingival plaque samples from 16 patients were negative for P. anaerobius DNA. None of the 43 pus samples analysed contained P. anaerobius DNA. These results suggest that P. anaerobius is not a major pathogen in adult periodontitis and dento-alveolar abscesses. The PCR assay is a more rapid, sensitive and specific alternative to culture-based methods for identification of P. anaerobius in clinical samples.

Specific PCR detection of Peptostreptococcus magnus.

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3' ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.