Quantification of Porphyromonas gingivalis and fimA genotypes in smoker chronic periodontitis

Teixeira SRL, Mattarazo F, Feres M, Figueiredo LC, de Faveri M, Simionato MRL, Mayer MPA. Quantification of Porphyromonas gingivalis and fimA genotypes in smoker chronic periodontitis. J Clin Periodontol 2009; 36: 482-487. doi: 10.1111/j.1600-051X.2009.01411.x. Porphyromonas gingivalis fimA genotypes were associated with virulence factors in vitro, but little evidence of an association with disease severity were shown in humans. We aimed to correlate levels of P. gingivalis fimA genotypes II and IV and probing depth in smoker-chronic periodontitis subjects. One hundred and sixty eight subgingival samples of 20 smokers non-treated chronic periodontitis subjects obtained from sites with different probing depths [shallow (<= 3 mm), intermediate (4-6 mm), deep (>= 7 mm)] were analysed by real-time PCR for P. gingivalis and genotypes fimA II and IV. P. gingivalis and fimA IV were detected in all subjects, whereas fimA II was detected in 18 subjects (90%). One hundred and fifty two sites (90.5%) harboured P. gingivalis. Genotypes II and IV were detected in 28% and 69.6% of sites, respectively. The proportions of genotypes II and IV in relation to P. gingivalis levels were similar in shallow, intermediate and deep probing sites (2.4%, 4.6%, 1.4% for genotype II and 15.5%, 17.7%, 11.7% for genotype IV, respectively), indicating that other non-tested genotypes were more abundant. Increased levels of genotype IV were associated with increasing probing depth, but not of genotype II. The data suggested an association between P. gingivalis genotype fimA IV and disease severity in smoker-chronic periodontitis subjects.

Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum-periodonticum-simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60-100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0%, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum-periodonticum-simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis; patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.

Reactive nitrogen intermediate susceptibility of Mycobacterium tuberculosis genotypes in an urban setting

BACKGROUND: Mycobacterium tuberculosis genotypes resistant to reactive nitrogen intermediates (RNI) predominate in certain urban communities, suggesting that this phenotype influences disease transmission. OBJECTIVE: To compare different M. tuberculosis genotypes for resistance to RNI generated in vitro. DESIGN: We genotyped 420 M. tuberculosis isolates from a neighborhood in Sao Paulo, Brazil, and analyzed them for susceptibility to RNI generated in acidified sodium nitrite (ASN) solution. RESULTS: Seventy-one (43%) of 167 recent-infection strains and 68 (43%) of 158 endogenous infection strains showed moderate- to high-level ASN resistance. CONCLUSION: ASN resistance of M. tuberculosis is not necessarily a determining factor for enhanced transmission.

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX monomodular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

An interleukin-1 beta (IL-1 beta) single-nucleotide polymorphism at position 3954 and red complex periodontopathogens independently and additively modulate the levels of IL-1 beta in diseased periodontal tissues

Inflammatory cytokines such as interieukin-1 beta (IL-1 beta) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-10 mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-10 promoter single-nucleotide polymorphism at position 3954 [IL-1 beta(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1 beta levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects in = 175) and the possible correlations with the clinical parameters of the disease. IL-1 beta(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1 beta levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1 beta(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1 beta (3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1 beta levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1 beta(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1 beta in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1 beta(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1 beta in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Tumor necrosis factor-alpha-308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased periodontal tissues

Background and Objective: Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). Material and Methods: The TNFA-308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. Results: No statistically significant differences were found in the frequency of the TNFA-308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA-308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA-308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. Conclusion: Our results demonstrate that the TNFA-308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome.

High genetic diversity in field isolates of Trypanosoma theileri assessed by analysis of cathepsin L-like sequences disclosed multiple and new genotypes infecting cattle in Thailand

In this study, we describe the first survey in Thailand of Trypanosoma theileri, a widespread and prevalent parasite of cattle that is transmitted by tabanid flies. Investigation of 210 bovine blood samples of Thai cattle from six farms by hematocrit centrifuge technique (HCT) revealed 14 samples with trypanosomes morphologically compatible to T. theileri. Additional animals were positive for T. theileri by PCR based on the Cathepsin L-like sequence (TthCATL-PCR) despite negative by HCT, indicating cryptic infections. Results revealed a prevalence of 26 +/- 15% (95% CI) of T. theileri infection. Additionally, 12 samples positive for T. theileri were detected in cattle from other 11 farms. From a total of 30 blood samples positive by HCT and/or PCR from 17 farms, seven were characterized to evaluate the genetic polymorphism of T. theileri through sequence analysis of PCR-amplified CATL DNA sequences. All CATL sequences of T. theileri from Thai cattle clustered with sequences of the previously described phylogenetic lineages TthI and TthII, supporting only two major lineages of T. theileri in cattle around the world. However, 11 of the 29 CATL sequences analyzed showed to be different, disclosing an unexpectedly large polymorphic genetic repertoire, with multiple genotypes of T. theileri not previously described in other countries circulating in Thai cattle. (C) 2011 Elsevier B.V. All rights reserved.

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FELLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main chide of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Characterization of spliced leader genes of Trypanosoma (Megatrypanum) theileri: phylogeographical analysis of Brazilian isolates from cattle supports spatial clustering of genotypes and parity with ribosomal markers

Trypanosoma (Megatrypanum) theileri from cattle and trypanosomes of other artiodactyls form a clade of closely related species in analyses using ribosomal sequences. Analysis of polymorphic sequences of a larger number of trypanosomes from broader geographical origins is required to evaluate the Clustering of isolates as suggested by previous studies. Here, we determined the sequences of the spliced leader (SL) genes of 21 isolates from cattle and 2 from water buffalo from distant regions of Brazil. Analysis of SL gene repeats revealed that the 5S rRNA gene is inserted within the intergenic region. Phylogeographical patterns inferred using SL sequences showed at least 5 major genotypes of T. theileri distributed in 2 strongly divergent lineages. Lineage TthI comprises genotypes IA and IB from buffalo and cattle, respectively, from the Southeast and Central regions, whereas genotype IC is restricted to cattle from the Southern region. Lineage Tth II includes cattle genotypes IIA, which is restricted to the North and Northeast, and IIB, found in the Centre, West, North and Northeast. PCR-RFLP of SL genes revealed valuable markers for genotyping T. theileri. The results of this study emphasize the genetic complexity and corroborate the geographical structuring of T. theileri genotypes found in cattle.

Infection rates and genotypes of Trypanosoma rangeli and T. cruzi infecting free-ranging Saguinus bicolor (Callitrichidae), a critically endangered primate of the Amazon Rainforest

Parasites of wild primates are important for conservation biology and human health due to their high potential to infect humans. In the Amazon region, non-human primates are commonly infected by Trypanosoma cruzi and T rangeli, which are also infective to man and several mammals. This is the first survey of trypanosomiasis in a critically endangered species of tamarin, Saguinus bicolor (Callitrichidae), from the Brazilian Amazon Rainforest. Of the 96 free-ranging specimens of S. bicolor examined 45 (46.8%) yielded blood smears positive for trypanosomes. T rangeli was detected in blood smears of 38 monkeys (39.6%) whereas T. cruzi was never detected. Seven animals (7.3%) presented trypanosomes of the subgenus Megatrypanum. Hemocultures detected 84 positive tamarins (87.5%). Seventy-two of 84 (85.7%) were morphologically diagnosed as T rangeli and 3 (3.1%) as T. cruzi. Nine tamarins (9.4%) yielded mixed cultures of these two species, which after successive passages generated six cultures exclusively of T. cruzi and two of T rangeli, with only one culture remaining mixed. Of the 72 cultures positive for T rangeli, 62 remained as established cultures and were genotyped: 8 were assigned to phylogenetic lineage A (12.9%) and 54 to lineage B (87.1%). Ten established cultures of T. cruzi were genotyped as TCI lineage (100%). Transmission of both trypanosome species, their potential risk to this endangered species and the role of wild primates as reservoirs for trypanosomes infective to humans are discussed. (C) 2008 Elsevier B.V. All rights reserved.