Effects of short chain fatty acids on effector mechanisms of neutrophils

Short chain fatty acids (SCFAs) are metabolic by products of anerobic bacteria fermentation. These fatty acids, despite being an important fuel for colonocytes, are also modulators of leukocyte function. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate, and butyrate) on function of neutrophils, and the possible mechanisms involved. Neutrophils obtained from rats by intraperitoneal lavage 4 h after injection of oyster glycogen solution (1%) were treated with non toxic concentrations of the fatty acids. After that, the following measurements were performed: phagocytosis and destruction of Candida albicans, production of ROS (O(2)(center dot-), H(2)O(2), and HOCl) and degranulation. Gene expression (p47(phox) and p22(phox)) and protein phosphorylation (p47(phox)) were analyzed by real time reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. Butyrate inhibited phagocytosis and killing of C. albicans. This SCFA also had an inhibitory effect on production of O(2)(center dot-), H(2)O(2), and HOCI by neutrophils stimulated with PMA or fMLP. This effect of butyrate was not caused by modulation of expression of NADPH oxidase subunits (p47(phox) and p22(phox)) but it was in part due to reduced levels of p47(phox) phosphorylation and an increase in the concentration of cyclic AMP. Acetate increased the production of O(2)(center dot-) and H(2)O(2), in the absence of stimuli but had no effect on phagocytosis and killing of C. albicans. Propionate had no effect on the parameters studied. These results suggest that butyrate can modulate neutrophil function, and thus could be important in inflammatory neutrophil-associated diseases. Copyright (C) 2008 John Wiley & Sons, Ltd.

Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis

Background/aim: The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with generalized aggressive periodontitis by using culture-independent molecular methods based on 16S ribosomal DNA cloning. Methods: Samples from 10 subjects with generalized aggressive periodontitis were selected. DNA was extracted and the 16S rRNA gene was amplified with the universal primer pairs 9F and 1525R. Amplified genes were cloned, sequenced, and identified by comparison with known 16S rRNA sequences. Results: One hundred and ten species were identified from 10 subjects and 1007 clones were sequenced. Of these, 70 species were most prevalent. Fifty-seven percent of the clone (40 taxa) sequences represented phylotypes for which no cultivated isolates have been reported. Several species of Selenomonas and Streptococcus were found at high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were formed by these two genera. Selenomonas sputigena, the species most commonly detected, was found in nine of 10 subjects. Other species of Selenomonas were often present at high levels, including S. noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas FT050, Selenomonas sp. P2PA_80, and Selenomonas sp. strain GAA14. The classical putative periodontal pathogens, such as, Aggregatibacter actinomycetemcomitans, was below the limit of detection and was not detected. Conclusion: These data suggest that other species, notably species of Selenomonas, may be associated with disease in generalized aggressive periodontitis subjects.

Presence of periodontopathic bacteria in coronary arteries from patients with chronic periodontitis

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries. (C) 2010 Elsevier Ltd. All rights reserved.

Hospital-based Surveillance to Evaluate the Impact of Rotavirus Vaccination in Sao Paulo, Brazil

Background: Brazil implemented routine immunization with the human rotavirus vaccine, Rotarix, in 2006 and vaccination coverage reached 81% in 2008 in Sao Paulo. Our aim was to assess the impact of immunization on the incidence of severe rotavirus acute gastroenteritis (AGE). Methods: We performed a 5-year (2004-2008) prospective surveillance at a sentinel hospital in Sao Paulo, with routine testing for rotavirus in all children less than 5 years of age hospitalized with AGE. Genotypes of positive samples were determined by reverse transcription polymerase chain reaction. Results: During the study, 655 children hospitalized with AGE were enrolled; of whom 169 (25.8%) were positive for rotavirus. In the post-vaccine period, a 59% reduction in the number of hospitalizations of rotavirus AGE and a 42.2% (95% confidence interval [CI], 18.6%-59.0%; P = 0.001) reduction in the proportion of rotavirus-positive results among children younger than 5 years were observed, with the greatest decline among infants (69.2%; 95% CI, 24.7%-87.4%; P = 0.004). Furthermore, the number of all-cause hospitalizations for AGE was reduced by 29% among children aged <5 years. The onset and peak incidences of rotavirus AGE occurred 3 months later in the 2007 and 2008 seasons compared with previous years. Genotype G2 accounted for 15%, 70%, and 100% of all cases identified, respectively, in 2006, 2007, and 2008. Conclusions: After vaccine implementation, a marked decline in rotavirus AGE hospitalizations was demonstrated among children younger than 5 years of age, with the greatest reduction in the age groups targeted for vaccination. The predominance of genotype G2P[4] highlights the need of continued postlicensure surveillance studies.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

OBJECTIVES To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil. METHODS Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus. RESULTS Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed. CONCLUSIONS Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.

Characterization of Paracoccidioides brasiliensis COX9, COX12, and COX16 respiratory genes

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that is the causative agent of paracoccidioidomyicosis (PCM), a human systemic granulomatous mycosis found in Latin America. Dimorphic transition from mycelium to yeast is required for establishing pathogenicity. Dimorphism is marked by changes in mitochondrial physiology, including modulation of respiration rate. In this work, we present the identification of three P. brasiliensis nuclear genes PbCOX9, PbCOX12, and PbCOX16 that code for structural sub-units and a putative assembly facilitator (PbCOX16) of the mitochondrial cytochrome c oxidase (COX), the terminal enzyme complex of the respiratory chain. We measured their expression pattern during the dimorphic transition from mycelium to yeast and back by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). Our results show that messages from these genes increase during the mycelium to yeast transition and decrease during the opposite conversion. This result supports active mitochondrial participation in the transition. Heterologous complementation of the corresponding Saccharomyces cerevisiae null mutant with the PbCOX9 gene was successfully obtained. (C) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Detection of Mycoplasma pulmonis in laboratory rats and technicians

Five species of mycoplasma are associated with several rat diseases. Mycoplasma pulmonis is the most important and most studied, possibly causing disease in rats and undermining the validity of laboratory experiments. M. pulmonis was isolated in 144/240 laboratory rats and identified by PCR in 155/240. This species was also detected in 12 human individuals (technicians of a laboratory animal house hold) in contact with these rats. The results were confirmed by sequencing of DNA products. Mycoplasma species are host specific; however, M. pulmonis was identified in humans, suggesting a case of unspecific colonization. Statistical analysis shows a greater risk for M. pulmonis colonizing individuals who are exposed to infected rats in animal facilities than individuals who do not. The detection of M. pulmonis in humans indicates a new status for this mollicute mycoplasmas in animal-holding facilities.

Intraspecific variation in 16S rRNA gene of Mycoplasma synoviae determined by DNA sequencing

Mycoplasma synoviae (MS) is an important avian pathogen may cause both respiratory disease and joint inflammation synovitis in poultry, causing economic losses to the Brazilian poultry industry. The genotypic variation in 16S rRNA gene is unknown. Partial sequences of 16S rRNA gene of 19 strains of M. synoviae were sequenced and analyzed in order to obtain molecular characterization and evaluation of the genetic variability of strains from distinct Brazilian areas of poultry production. Different polymorphic patterns were observed. The number of polymorphic alterations in the studied strains ranged from 0 to 6. The nucleotide variations, including deletion, insertion and substitutions, ranged from 3 to 5. The genotypic diversity observed in this study may be explained by spontaneous mutations that may occur when a lineage remains in the same flock for long periods. The culling and reposition in poultry flocks may be responsible for the entry of new strains in different areas. (C) 2008 Elsevier Ltd. All rights reserved.

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human P-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully. (c) 2007 Elsevier B.V. All rights reserved.

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.

Avaliação do potencial de formigas (Hymenoptera: formicidae) como vetores mecânicos de bactérias do gêneroSstaphylococcus no ambiente hospitalar.

In a hospital environment, these bacteria can be spread by insects such as ants, which are characterized by high adaptability to the urban environment. Staphylococcus is a leading cause of hospital infection. In Europe, Latin America, USA and Canada, the group of coagulase negative staphylococci (CoNS) is the second leading cause of these infections, according to SENTRY (antimicrobial surveillance program- EUA). In this study, we investigated the potential of ants (Hymenoptera: Formicidae) as vehicle mechanics of Staphylococcus bacteria in a public hospital, in Natal-RN. The ants were collected, day and night, from June 2007 to may 2008, in the following sectors: hospitals, laundry, kitchen, blood bank. The ants were identified according to the identification key of Bolton, 1997. For the analysis of staphylococci, the ants were incubated in broth Tryptic Soy Broth (TSB) for 24 hours at 35 º C and then incubated on Mannitol Salt Agar. The typical colonies of staphylococci incubated for 24 hours at 35 ° C in Tryptic Soy Agar for the characterization tests (Gram stain, catalase, susceptibility to bacitracin and free coagulase). The identification of CoNS was performed through biochemical tests: susceptibility to novobiocin, growth under anaerobic conditions, presence of urease, the ornithine decarboxylation and acid production from the sugars mannose, maltose, trehalose, mannitol and xylose. The antimicrobial susceptibility examined by disk-diffusion technique. The technique of Polymerase Chain Reaction was used to confirm the presence of mecA gene and the ability to produce biofilm was verified by testing in vitro using polystyrene inert surface, in samples of resistant staphylococci. Among 440 ants, 85 (19.1%) were carrying coagulase-negative staphylococci (CoNS) of the species Staphylococcus saprophyticus (17), Staphylococcus epidermidis (15), Staphylococcus xylosus (13), Staphylococcus hominis hominis (10), Staphylococcus lugdunensis (10), Staphylococcus warneri (6), Staphylococcus cohnii urealyticum (5), Staphylococcus haemolyticus (3), Staphylococcus simulans (3), Staphylococcus cohnii cohnii (2), and Staphylococcus capitis (1). No Staphylococcus aureus was found. Among the isolates, 30.58% showed resistance to erythromycin. Two samples of CoNS (2.35%), obtained from the ant Tapinoma melanocephalum collected in the post-surgical female ward, S. Hominis hominis and S. lugdunensis harbored the mecA gene and were resistant to multiple antibiotics, and the specie S. hominis hominis even showed to be a biofilm producer. This study proves that ants act as carriers of multidrug-resistant coagulase-negative Staphylococci and biofilm producers and points to the risk of the spreading of pathogenic microorganisms by this insect in the hospital environment

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

Análise da frequência alélica de 15 LOCI STR na população do Rio Grande do Norte

Human population have a significant number of polymorphic loci, whose use and applications range from construction of linkage maps, to study the evolution of populations, through the determination of paternity, forensic medicine and migration. Currently, STRs (Short Tanden Repeats) markers are considered the major markers for human identification, mainly due to its abundance and high variability because of the fact that they are easily amplifiable by PCR (Polymerase Chain Reaction), work with low amounts of DNA and be capable of automation processes involving fluorescence detection. The creation of regional databases containing allele frequencies of population provide subsidies to increase the reliability of the results of determining the genetic link. This paper aims to obtain a database of allele frequencies of 15 polymorphic molecular loci (D8S1179, D21S11, D7S820, CSF1PO, D19S433, vWA, TPOX, D18S51, D3S1358, TH01, D13S317, D16S539, D2S1338, D5S818 e FGA) in a population classifies as born in the State of Rio Grande do Norte, Brazil, totaling 1100 unrelated individuals. To evaluate the frequency, DNA samples were submitted to PCR amplification, followed by capilarry electrophoresis genetic sequencer. The frequencies identified in this study were compared with brazilian population in general and other states in Brazil. Except for the loci D21S11, D19S433 and D2D1338, the genotypes found were in Hardy-Weinberg equilibrium and no significant differences among the frequencies were found in the populations studied. The most informative loci was D2S1338 and D18S51, and the less informative is the locus TPOX

Development of Y-chromosome-Specific SCAR Markers Conserved in Taurine, Zebu and Bubaline Cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.