Detection of Viruses from Bioaerosols Using Anion Exchange Resin.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

Colorimetric paper-based detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes of agricultural water.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Evaluation of modified moore swabs and continuous flow centrifugation for concentration of Salmonella and Escherichia coli O157:H7 from large volumes of water.

Modified Moore swabs (MMS; consisting of a polyvinyl chloride cartridge filled with gauze) capture microorganisms within the packed gauze as water flows through the cartridge, while continuous flow centrifugation (CFC) uses centrifugation to sediment the microorganisms while water continuously flows in the system. This study evaluated and compared the efficacy of MMS and CFC for concentration and subsequent detection of Escherichia coli O157:H7 and Salmonella from large volumes of water (10 liters). Water samples were spiked at levels of 10(1), 10(2), 10(3), and 10(4) CFU/100 ml with three-strain cocktails of either E. coli O157:H7 or Salmonella serovars, which had been previously transformed with a plasmid to express resistance to ampicillin as well as green, red, or cyan fluorescent proteins. Plating was performed before and after concentration on tryptic soy agar supplemented with ampicillin in order to quantitate the concentration efficiencies of each method. The two lowest spiking levels were also enriched in low volumes of tryptic soy broth supplemented with ampicillin followed by testing via lateral flow devices. Significant (P < 0.05) concentrations of initial levels of E. coli O157:H7 in the range of 0.7 to 1.0 and 1.2 to 1.4 log were achieved within approximately 35 min of processing time via MMS and CFC, respectively. Similarly, significant (P < 0.05) concentrations were also achieved for Salmonella with 0.9 to 1.2 and 1.2 to 1.4 log concentration for MMS and CFC, respectively. There were no statistical differences (P > 0.05) between the two concentration methods in their ability to concentrate either of the two target bacteria. Significantly (P > 0.05) more spiked samples were detected by lateral flow devices following concentration and enrichment than for nonconcentrated, enriched samples. It is concluded that both MMS and CFC have potential to be used to enhance the sensitivity of downstream bacterial detection methods used to test irrigation water for the presence of foodborne pathogens.

Secreted proteins of Avibacterium paragallinarum are lethal for chicken embryo.

Avibacterium paragallinarum causes infectious coryza in chickens. This bacterium secretes proteins of 110 kDa (a putative RTX protein) and 120 kDa. Expression of these proteins increases by the addition of CaCl(2), MgSO(4), MnSO(4), or ferric ammonium citrate and diminishes with CuSO(4) or ZnCl(2). Protein expression is optimal at 37 degrees C and pH 7.5. Mortality (90-100%) of chicken embryos was observed when secreted proteins (SPs) from A. paragallinarum reference or field isolates (serogroup A or C) were inoculated via yolk sac and was not observed when SPs from A. avium, a chicken respiratory tract indigenous bacterium, were inoculated. A. paragallinarum SPs could contain toxins responsible for the embryo deaths. Indeed, presence of the putative RTX protein of 110 kDa was confirmed by Western blotting with antibodies against the Actinobacillus pleuropneumoniae RTX ApxI, a closely related RTX protein.

Membrane vesicles released by Avibacterium paragallinarum contain putative virulence factors.

Avibacterium paragallinarum, the causative agent of infectious coryza, releases extracellular membrane vesicles (MVs), containing immunogenic proteins, proteases, putative RTX proteins, haemagglutinin, and nucleic acids, into the medium. MVs ranging 50-300 nm in diameter were observed by electron microscopy. They contained immunogenic proteins in the range of 20-160 kDa, detected using vaccinated or experimentally infected chicken sera raised against Av. paragallinarum, but not in pooled sera from specific pathogen-free chickens. Proteolytic activity was not detected in MVs through zymograms; however, immune recognition of high molecular mass bands was observed by Western blotting using an antiprotease serum against Actinobacillus pleuropneumoniae serotype 1 purified protease, suggesting its presence. MVs agglutinated glutaraldehyde-fixed chicken red blood cells indicating the presence of haemagglutinating antigens. Nucleic acids were also detected inside MVs. Avibacterium paragallinarum releases MVs containing putative virulence factors, which could be important in the pathogenesis of infectious coryza.

Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein.

Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.