The applicability of hns and fimA primers for detecting Salmonella in bioaerosols associated with animal and municipal wastes

The ability to rapidly screen environmental samples for specific pathogens such as Salmonella spp., is of particular importance in molecular epidemiology. Although gene amplification reactions allow the rapid detection of microorganisms, the use of appropriate oligonucleotide primers targeted against specific microbial genes is critical for accurate detection specificity and sensitivity. Primers such as fimA and hns have been previously shown to be specific for pure cultures of Salmonella. However, the analysis of environmental samples requires post-amplification hybridizations to detect amplicons, since the presence of inhibitory environmental components reduces amplification efficiency of the target organism. These sensitive post-amplification approaches also enable the detection of spurious amplification from non-target sequences. Bioaerosols associated with animal facilities and municipal wastes contain a diverse array of pathogens including Clostridium spp. In our studies, hybridization sensor data revealed spurious amplification of clostridial species with Salmonella hns primers. Specificity checks using type cultures of Clostridium spp. revealed non-specific amplification by hns primers. These results suggest that fimA primers may be better suited to screen Salmonella-specific sequences in environmental samples, especially those obtained from animal and municipal waste facilities.     

1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation

Human foreskin keratinocytes in culture produce 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) from 25-hydroxycholecalciferol (25-(OH)D3). The production of 1,25-(OH)2D3 by these cells correlated with the early events of differentiation such as expression of transglutaminase activity and the levels of a precursor protein for the cornified envelopes, involucrin. In contrast, the increased production of 24,25-(OH)2D3, as 1,25-(OH)2D3 production declined, correlated with the terminal differentiation marker, cornified envelope formation. Exogenous 1,25-(OH)2D3 (10(-11)-10(-9) M) inhibited the 1-alpha-hydroxylase at all stages of growth of these cells. Keratinocytes in culture expressed receptors for 1,25-(OH)2D3 which had similar sedimentation behavior in sucrose density gradients as chick intestinal cytosol receptors. Cells in early stages of growth (preconfluent and confluent) contained higher numbers of receptors (26-27 fmol/mg protein) than post-confluent cells. The dissociation constant (237-278 pM) of these receptors for 1,25-(OH)2D3 was not consistently altered by differentiation. Since 1,25-(OH)2D3 is a potent stimulator of cell differentiation in a variety of systems including the epidermis, our results suggest the possibility that endogenous 1,25-(OH)2D3 production may participate in the differentiation of keratinocytes in culture and, perhaps, in vivo.     

Rôle of male accessory gland substance in the regulation of blood intake by mosquitoes

The presence of male accessory gland substance during mating is shown to increase the blood intake in the adult female Aedes aegypti and Culex pipiens fatigans. Females mated with males whose accessory glands were surgically removed took significantly less blood than females mated with normal males. However, females mated with males whose seminal vesicles were surgically removed took as much blood as the controls.     

Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS-TTEGDA support for the investigation of HCV infection in human blood.

Four peptides were designed and synthesized on a highly solvating copolymer of tetraethyleneglycol diacrylate cross-linked polystyrene (PS-TTEGDA) support with very high purity and yield. The polymer was synthesized in various cross-linking densities (1, 2, 3, 4, 5 and 10%) using radical aqueous suspension polymerization. Four per cent PS-TTEGDA resin showed rigidity and mechanical characteristics comparable with those of divinylbenzene cross-linked polystyrene (PS-DVB) support. Swelling and solvation characteristics of PS-TTEGDA were much higher than PS-DVB support in all solvents used in solid-phase peptide synthesis. Forty-eight hour treatment of the support with neat trifluoroacetic acid did not show any change in its infrared spectra. PS-TTEGDA could be functionalized with chloromethyl, aminomethyl and hydroxymethyl functional groups under various controlled conditions. Synthetic utility of the support was demonstrated by the synthesis of four peptides selected from the envelope and nonstructural protein region of the prototype hepatitis C virus (HCV). These peptides were later used successfully to develop a peptide-based immunoassay (PBEIA) for the detection of HCV immunity. Peptides designed from the NS1 and NS4 protein regions were found to be very promising for the development of a new diagnostic kit to detect HCV infection in human blood. Peptide purity was tested by RP-FPLC and the peptide identity was confirmed by amino acid analysis.     

Identification of Cryptosporidium parvum Oocysts by an Artificial Neural Network Approach

Microscopic detection of Cryptosporidium parvum oocysts is time-consuming, requires trained analysts, and is frequently subject to significant human errors. Artificial neural networks (ANN) were developed to help identify immunofluorescently labeled C. parvum oocysts. A total of 525 digitized images of immunofluorescently labeled oocysts, fluorescent microspheres, and other miscellaneous nonoocyst images were employed in the training of the ANN. The images were cropped to a 36- by 36-pixel image, and the cropped images were placed into two categories, oocyst and nonoocyst images. The images were converted to grayscale and processed into a histogram of gray color pixel intensity. Commercially available software was used to develop and train the ANN. The networks were optimized by varying the number of training images, number of hidden neurons, and a combination of these two parameters. The network performance was then evaluated using a set of 362 unique testing images which the network had never “seen” before. Under optimized conditions, the correct identification of authentic oocyst images ranged from 81 to 97%, and the correct identification of nonoocyst images ranged from 78 to 82%, depending on the type of fluorescent antibody that was employed. The results indicate that the ANN developed were able to generalize the training images and subsequently discern previously unseen oocyst images efficiently and reproducibly. Thus, ANN can be used to reduce human errors associated with the microscopic detection of Cryptosporidium oocysts.     

Coelomomyces opifexi (Pillai & Smith). Coelomomycetaceae: Blastocladiales II. Experiments in sporangial germination

Summary Laboratory experiments on sporangial germination and zoospore activity in Coelomomyces opifexi which utilises a suparlittoral environment are described. Sporangial germination depends upon (a) salinity of the medium used and (b) whether the sporangia were derived from living or deceased larvae. Sporangia from living larvae germinated almost instantaneously in distilled, tap, brackish pond and sea water with a salinity of 4.2 There was only partial germination at a salinity of 17, and none at all in 35 (full sea water). Sporangia from deceased larvae required a conditioning of 7 days or more under moisture at 23°C or 28°C before germination. Sporangia from living and moribund larvae became thick-walled and darker when exposed to a salinity of 8.5 or higher. These, likewise, required a conditioning period for germination. The biological and ecological significance of these observations are discussed.

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Zusammenfassung Die in vitro-Versuche an das Keimen und die Zootätigkeit von der supralitteral lebenden spezies Coelomomyces opifexi sind hier beschrieben worden. Das sporenbeheltische Keimen ist abhängig von (a) der Salzhaltigkeit der Umgebung und (b) ob die Sporenbehalter von lebenden oder toten Puppen erhalten sind. Die von lebenden Puppen herstammenden Sporenbehalter keimen sofort in distilliertem, Leitings,- Brack- und 4.2 tigem Salzwasser, nur zum Teil in 17 tigem Salzwasser und gar nicht in Meereswasser (35). Die Wänder der von toten Puppen herstammende Sporenbehalter waren dichter und schwarzer und brauchten mindestens 7 Tage zum keimen in einer feuchten Umgebung von 23° bis zu 28°C. Wenn der Salzgehalt stieg über 8.5, so worden die Wänder beider Arten dichter und schwarzer und brauchten ebenso eine bedingte Periode zum keimen. Die biologische und ekologische Bedeutung dieser Beobachtungen sind diskutiert worden.