Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.
Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.Several Cryptosporidium species can cause severe acute diarrhea in humans and animals (5, 6, 13). Human cryptosporidiosis is usually self-resolving within a few days, but immunocompromised patients can develop life-threatening complications. Outbreaks due to drinking water contamination continue to occur (7,10,18,32), and there is no effective treatment, making cryptosporidiosis a major public health issue and economic problem. Diagnosis is generally based on microscopic detection of oocysts in stools, but this offers no information on the infecting species and is not suited to epidemiological investigations.The following 13 Cryptosporidium species are currently accepted, on the basis of host specificity, pathogenesis, morphology (9) and genotyping (8,16,21): Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium wrairi, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium andersoni, and Cryptosporidium muris as infecting mammals; Cryptosporidium baileyi, Cryptosporidium meleagridis, and Cryptosporidium galli as infecting birds; Cryptosporidium serpentis and Cryptosporidium saurophilum as infecting reptiles; and Cryptosporidium molnari as infecting fish (36). Recent phylogenetic analyses based on sequencing of the small subunit rRNA gene (18S rRNA) (21,22,24,33,34), the hsp 70 gene (31), or other housekeeping or structural genes (24,(28)(29)(30) show a complex multispecies organization of the g...
Accidental ingestion of natural waters while bathing carries a risk of infection by waterborne protozoa such as Cryptosporidium, Giardia and, possibly, microsporidia. In order to evaluate this risk, we conducted a one-year prospective study of two recreational lakes and three river sites located near Paris, where bathing and boating are frequent. Twenty-litre water samples were collected monthly from each site. Concentrated samples were submitted to immunomagnetic separation followed by immunofluorescence (IMS-IF) for Cryptosporidium and Giardia detection. PCR and PCR restriction fragment length polymorphism (PCR-RFLP) were used for the genetic characterization of Cryptosporidium species on IMS-IF-positive samples. PCR were systematically performed to detect Enterocytozoon bieneusi. Bacteria counts were also determined. IMS-IF revealed low counts of Giardia cysts and Cryptosporidium oocysts in the recreational lakes, with occasional peaks (max. 165 cysts/10 L and 9 oocysts/10 L). By contrast, the river sites were consistently and sometimes heavily contaminated throughout the year. Enterocytozoon bieneusi was found in only two river samples. PCR-RFLP genotyping showed the presence of C. hominis and C. parvum. No correlation was found between the presence or counts of parasites and bacteria, except between the presence of Giardia and high counts of Escherichia coli and enterococci. Based on a previously developed model for quantitative risk assessment of waterborne parasitic infections, we estimated that the mean risk of infection by Cryptosporidium and Giardia associated with swimming was <10(-4) in the recreational lakes, and frequently higher at the river sites.
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