A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field-collected Anopheles mosquitoes.

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field-collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real-time polymerase chain reaction (RT-PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme-linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP-ELISA). However, because of the relatively high costs associated with equipment and reagents, RT-PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA-based nested PCR protocol, modified from a loop-mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP-ELISAs in field-collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4-fold higher sensitivity than the CSP-ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium-positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.

Influence of a preschool preventive dental programme on caries prevalence, oral care and secretory immunity to Streptococcus mutans in young adults.

OBJECTIVE: To evaluate oral hygiene habits, decayed, missing and filled teeth (DMFT) and surfaces (DMFS), dental care, dietetic habits and anti-Streptococcus mutans salivary secretory Immunoglobulin A (SIgA) in young adults who attended a preventive programme during preschool age. MATERIAL AND METHODS: The study group (Baby Clinic) comprised 72 patients, aged 18-25 years, who had participated in the Baby Clinic preventive programme. The control group was age- and gender-matched. The patients were examined and unstimulated whole saliva was sampled for detection of anti-S. mutansSIgA antibodies. RESULTS: Control patients presented increased DMFS scores (P < .05). Hygiene habits, cariogenic diet and antibody levels were not different between groups (P > .05). Baby Clinic patients presented better periodontal status (P < .005), less calculus (P < .005) and bleeding on probing (P < .005), and reported visiting dental services more regularly (P < .05). Adjusted multivariate linear regression analysis demonstrated that DMFT was associated with study group (P < .05), gender (P < .05), parents' education (P < .05), carbohydrate intake (P < .001) and levels of anti-S. mutansSIgA (P < .007). DMFS was associated with time elapsed since the last visit to the dentist (P < .005) and weekly carbohydrate intake (P < .005). CONCLUSION: Preventive programmes for preschool children positively impact on DMFS and periodontal status in young adults, but have no long-term effects on dietary or hygiene habits.

Isolation and molecular identification of lactic acid bacteria and Bifidobacterium spp. from faeces of the blue-fronted Amazon parrot in Brazil.

In Brazil, the blue-fronted Amazon parrot (Amazona aestiva) is a common pet. The faecal microbiota of these birds include a wide variety of bacterial species, the majority of which belong to the Gram-positive lactic acid bacteria (LAB) clade. The aim of this study was to investigate differences in the diversity and abundance of LAB and Bifidobacterium spp. in the cloacae between wild and captive birds and to select, identify and characterise LAB for consideration as a parrot probiotic. Cloacal swabs were collected from 26 wild and 26 captive birds. Bacterial DNA was extracted, and the 16S rRNA genes were amplified. The numbers of PCR-positive Enterococcus, Pediococcus, and Lactobacillus species isolated from wild and captive birds were significantly different (P<0.05). Enterococcus was the most frequently isolated genus, followed by Pediococcus, Lactobacillus, Lactococcus and Bifidobacterium. Enterococcus faecium, Pediococcus pentosaceus, Lactococcus lactis, Lactobacillus coryniformis, Lactobacillus sanfranciscensis and Bifidobacterium bifidum were the most frequently isolated species from all birds. This study increases our understanding of the faecal microbiota, and may help to improve the nutrition and habitat management of captive and wild parrots. The bacterial population identified in the faecal microbiota of clinically healthy wild and captive parrots can serve as a database to analyse variations in the gut microbiota of pathogen-infected parrots and to develop probiotics specific to these genera.

Ongoing outbreak of dengue type 1 in the Autonomous Region of Madeira, Portugal: preliminary report.

Following the identification of two autochthonous cases of dengue type 1 on 3 October 2012, an outbreak of dengue fever has been reported in Madeira, Portugal. As of 25 November, 1,891 cases have been detected on the island where the vector Aedes aegypti had been established in some areas since 2005. This event represents the first epidemic of dengue fever in Europe since 1928 and concerted control measures have been initiated by local health authorities.

Population genetic structure of the blue-fronted Amazon (Amazona aestiva, Psittacidae: Aves) based on nuclear microsatellite loci: implications for conservation.

The blue-fronted Amazon (Amazona aestiva) is a widely distributed Neotropical parrot and one of the most captured parrots in nature to supply the illegal trade of wild animals. The objectives of the present study were to analyze the genetic structure of A. aestiva to identify management units and support conservation planning and to verified if A. aestiva populations have undergone a recent bottleneck due to habitat loss and capture for the pet trade. The genetic structure was accessed by analyzing six microsatellite loci in 74 individuals of A. aestiva, including samples from the two subspecies (A. a. aestiva and A. a. xanthopteryx), from five populations: four in Brazil and one in Argentina. A significant genetic differentiation (theta = 0.007, p = 0.005) could be detected only between the most distant populations, Tocantins and Argentina, localized at the northeast and southwest limits of the sample sites, respectively. There was no evidence of inbreeding within or between populations, suggesting random mating among individuals. These results suggest a clinal distribution of genetic variability, as observed for variation in plumage color of the two A. aestiva subspecies. Bottleneck analysis did not show a recent reduction in population size. Thus, for the management and conservation of the species, the populations from Argentina and Tocantins should be considered as different management units, and the other populations from the center of the geographical distribution as another management unit.

Sodium fluoride is a less efficient human cell mutagen at low concentrations.

Sodium fluoride was found to induce gene-locus mutations at the thymidine kinase (tk) and hypoxanthine guanine phosphoribosyl transferase (hgprt) loci in human lymphoblastoid cells. A single, 28 hr exposure to up to 600 micrograms/ml sodium fluoride induced a concentration-dependent increase in mutant fraction at both gene loci and reduced cell survival to 12% relative to negative control cultures. When cells were exposed to sodium fluoride concentrations that were only minimally toxic using a 20 day treatment protocol, no detectable induction of mutation was ob-served at the hgprt locus, and induction of mutation was observed at the tk locus only for treatment with 65 micrograms/ml sodium fluoride; exposure to 50 and 35 micrograms/ml sodium fluoride did not induce detectable mutation. The assay protocol used was of sufficient statistical sensitivity to detect the level of mutation predicted based on a linear extrapolation of data obtained from a 28 hour exposure. The implications of these observations with regard to the extrapolability of mutagenicity data to low concentrations are discussed.

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line.

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.

Mutagenicity of 1,2-dichloroethane and 1,2-dibromoethane in two human lymphoblastoid cell lines.

1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were tested for the ability to induce gene mutations in two human lymphoblastoid cell lines, designated AHH-1 and TK6. Both chemicals were 'direct-acting' mutagens in both cell lines. DBE was essentially equally mutagenic in TK6 cells and AHH-1 cells. In contrast, DCE was 25-fold more mutagenic in the AHH-1 cell line than in the TK6 cell line. This differential sensitivity between AHH-1 cells and TK6 cells was related to the levels of glutathione S-transferase activity in these two cell lines.

The aza-arenes as mutagens for Salmonella typhimurium.

Several unsubstituted aza-arenes have been found to be more mutagenic to Salmonella typhimurium than their corresponding parent hydrocarbons. In most cases, the activity of these compounds depended on the presence of a post-mitochondrial supernatant for metabolic activation, although acridine was mutagenic only in the absence of such an activating system. An examination of the effect of the metabolizing system's concentration on mutagenicity showed that quinoline, benzo[f]quinoline, and phenanthridine have different optima. In an attempt to uncover active intermediates in aza-arene metabolism, N-oxides of quinoline and phenanthridine were synthesized and found to be non-mutagenic, and coincubation with the epoxide hydrase inhibitor trichloropropylene oxide did not affect the mutagenic activity of quinoline or phenanthridine.