Anaerobic Transfer of Antibiotic Resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 × 10⁻⁹ and 5.0 × 10⁻⁸ cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 × 10⁻⁷ and 6.5 × 10⁻⁸ cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

Efficacy of Electrolyzed Oxidizing Water for Inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes

The efficacy of electrolyzed oxidizing water for inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes was evaluated. A five-strain mixture of E. coli O157:H7, S. enteritidis, or L. monocytogenes of approximately 10⁸ CFU/ml was inoculated in 9 ml of electrolyzed oxidizing water (treatment) or 9 ml of sterile, deionized water (control) and incubated at 4 or 23°C for 0, 5, 10, and 15 min; at 35°C for 0, 2, 4, and 6 min; or at 45°C for 0, 1, 3, and 5 min. The surviving population of each pathogen at each sampling time was determined on tryptic soy agar. At 4 or 23°C, an exposure time of 5 min reduced the populations of all three pathogens in the treatment samples by approximately 7 log CFU/ml, with complete inactivation by 10 min of exposure. A reduction of ≥7 log CFU/ml in the levels of the three pathogens occurred in the treatment samples incubated for 1 min at 45°C or for 2 min at 35°C. The bacterial counts of all three pathogens in control samples remained the same throughout the incubation at all four temperatures. Results indicate that electrolyzed oxidizing water may be a useful disinfectant, but appropriate applications need to be validated.     

Solution Structure of Mouse Hepatitis Virus (MHV) nsp3a and Determinants of the Interaction with MHV Nucleocapsid (N) Protein

Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses that infect a variety of vertebrate hosts. The CoV nucleocapsid (N) protein contains two structurally independent RNA binding domains, designated the N-terminal domain (NTD) and the dimeric C-terminal domain (CTD), joined by a charged linker region rich in serine and arginine residues (SR-rich linker). An important goal in unraveling N function is to molecularly characterize N-protein interactions. Recent genetic evidence suggests that N interacts with nsp3a, a component of the viral replicase. Here we present the solution nuclear magnetic resonance (NMR) structure of mouse hepatitis virus (MHV) nsp3a and show, using isothermal titration calorimetry, that MHV N219, an N construct that extends into the SR-rich linker (residues 60 to 219), binds cognate nsp3a with high affinity (equilibrium association constant [K_a], [1.4 ± 0.3] × 10⁶ M⁻¹). In contrast, neither N197, an N construct containing only the folded NTD (residues 60 to 197), nor the CTD dimer (residues 260 to 380) binds nsp3a with detectable affinity. This indicates that the key nsp3a binding determinants localize to the SR-rich linker, a finding consistent with those of reverse genetics studies. NMR chemical shift perturbation analysis reveals that the N-terminal region of an MHV N SR-rich linker peptide (residues 198 to 230) binds to the acidic face of MHV nsp3a containing the acidic α2 helix with an affinity (expressed as K_a) of 8.1 × 10³ M⁻¹. These studies reveal that the SR-rich linker of MHV N is necessary but not sufficient to maintain this high-affinity binding to N.     

Inactivation of Kanamycin,Neomycin, and Streptomycin by Enzymes Obtained in Cells of Pseudomonas aeruginosa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 × g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3′-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

Electron Microscopy of Viral RNA: Molecular Weight Determination of Bacterial and Animal Virus RNAs

A method for preparation of single-stranded RNA for electron microscopy determination of molecular weight is reported. The method uses treatment with formaldehyde at elevated temperatures to remove secondary structure and spreading in a protein monolayer from 50% formamide onto a 50% formamide hypophase. Molecular weights were determined for some bacterial and animal viruses, for which conflicting values had been reported earlier. Molecular weights determined by the method, using Escherichia coli large subunit rRNA for a standard (1.1 × 10⁶), are as follows: E. coli small subunit rRNA, 0.53 × 10⁶; coliphage f2-RNA, 1.3 × 10⁶; Qβ-RNA, 1.55 × 10⁶; and Newcastle disease virus RNA, 5.78 × 10⁶.     

Effect of Hypochlorite-Based Disinfectants on Inactivation of Murine Norovirus and Attempt to Eliminate or Prevent Infection in Mice by Additionto Drinking Water

We evaluated the in vitro efficacy of weak acid hypochlorous solution (WAHS) against murine norovirus (MNV) by plaque assay and compared the efficacy with diluted NaOCl (Purelox) and 70% ethanol. WAHS was as effective as 70% ethanol and diluted Purelox for 0.5-min reactions. For 0.5-min reactions in the presence of mouse feces emulsion, the efficacy of WHAS and 1:600 diluted Purelox was decreased, reducing the virus titers by 2.3 and 2.6 log₁₀, respectively, while 70% ethanol reduced the titer by more than 5 log₁₀. However, WAHS showed more than 5 log₁₀ reductions for the 5-min reaction even in the presence of feces emulsion. Since WAHS showed enough efficacy in inactivating MNV in vitro, we tried to eliminate MNV from MNV-infected mice by substituting WAHS for their drinking water. However, MNV was found to be positive in feces of mice drinking WAHS by an RT-nested PCR and plaque assay. To investigate whether hypochlorite-based disinfectants could prevent infection of a mouse with MNV, WAHS or 1:6,000 diluted Purelox was substituted for the drinking water of mice for 2 or 4 weeks, and then the mice were placed in a cage with an MNV-infected mouse. The supply of disinfectants was continued after cohabitation, but MNV was detected in the feces of all the mice at 1 week after cohabitation. In this study, we tried to eliminate and prevent MNV infection from mice by supplying hypochlorite-based disinfectants as an easy and low-cost method. Unfortunately, drinking disinfectants was ineffective, so it is important to keep the facility environment clean by use of effective disinfectants. Also, animals introduced into facilities should be tested as MNV free by quarantine and periodically confirmed as MNV free by microbiological monitoring.     

Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 × 10³ CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 × 10⁴ or 2.8 × 10⁷ CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.     

Three-way interactions between mosquito population,viral strain and temperature underlying chikungunya virus transmission potential

Interactions between pathogens and their insect vectors in nature are under the control of both genetic and non-genetic factors, yet most studies on mosquito vector competence for human pathogens are conducted in laboratory systems that do not consider genetic and/or environmental variability. Evaluating the risk of emergence of arthropod-borne viruses (arboviruses) of public health importance such as chikungunya virus (CHIKV) requires a more realistic appraisal of genetic and environmental contributions to vector competence. In particular, sources of variation do not necessarily act independently and may combine in the form of interactions. Here, we measured CHIKV transmission potential by the mosquito Aedes albopictus in all combinations of six worldwide vector populations, two virus strains and two ambient temperatures (20°C and 28°C). Overall, CHIKV transmission potential by Ae. albopictus strongly depended on the three-way combination of mosquito population, virus strain and temperature. Such genotype-by-genotype-by-environment (G × G × E) interactions question the relevance of vector competence studies conducted with a simpler set of conditions. Our results highlight the need to account for the complex interplay between vectors, pathogens and environmental factors to accurately assess the potential of vector-borne diseases to emerge.     

Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10⁹ and 3.0 × 10⁹ mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques'' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.     

Physicochemical properties of tipula iridescent virus

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 × 10⁸, with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 ± 0.2%. At 260 mμ, the virus gave an uncorrected absorbance of 18.2 cm²/mg of virus and a light-scattering corrected absorbance of 9.8 cm²/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.     

Genome-wide Association Study Identifies TNFSF15 and POU2AF1 as Susceptibility Loci for Primary Biliary Cirrhosis in the Japanese Population

For the identification of susceptibility loci for primary biliary cirrhosis (PBC), a genome-wide association study (GWAS) was performed in 963 Japanese individuals (487 PBC cases and 476 healthy controls) and in a subsequent replication study that included 1,402 other Japanese individuals (787 cases and 615 controls). In addition to the most significant susceptibility region, human leukocyte antigen (HLA), we identified two significant susceptibility loci, TNFSF15 (rs4979462) and POU2AF1 (rs4938534) (combined odds ratio [OR] = 1.56, p = 2.84 × 10⁻¹⁴ for rs4979462, and combined OR = 1.39, p = 2.38 × 10⁻⁸ for rs4938534). Among 21 non-HLA susceptibility loci for PBC identified in GWASs of individuals of European descent, three loci (IL7R, IKZF3, and CD80) showed significant associations (combined p = 3.66 × 10⁻⁸, 3.66 × 10⁻⁹, and 3.04 × 10⁻⁹, respectively) and STAT4 and NFKB1 loci showed suggestive association with PBC (combined p = 1.11 × 10⁻⁶ and 1.42 × 10⁻⁷, respectively) in the Japanese population. These observations indicated the existence of ethnic differences in genetic susceptibility loci to PBC and the importance of TNF signaling and B cell differentiation for the development of PBC in individuals of European descent and Japanese individuals.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by gamma-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by γ-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Identification of Cyanophage Ma-LBP and Infection of the Cyanobacterium Microcystis aeruginosa from an Australian Subtropical Lake by the Virus

Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium Microcystis aeruginosa (host). M. aeruginosa was isolated, cultured, and characterized from a subtropical monomictic lake—Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the M. aeruginosa host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h⁻¹; generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of M. aeruginosa cell lysis (r² = 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the Podoviridiae group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 × 10⁴ cyanophage·ml⁻¹, representing 0.23% of the natural viral population of 2.46 × 10⁷·ml⁻¹. Our results showed that this cyanophage could be a major natural control mechanism of M. aeruginosa abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production.     

Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes

Previously, we showed that infecting human intestinal epithelial cells (Caco-2) with rotavirus (RV) increases the release of extracellular vesicles (EVs) with an immunomodulatory function that, upon concentration at 100,000×g, present buoyant densities on a sucrose gradient of between 1.10 to 1.18 g/ml (characteristic of exosomes) and higher than 1.24 g/ml (proposed for apoptotic bodies). The effect of cellular death induced by RV on the composition of these EV is unknown. Here, we evaluated exosome (CD63, Hsc70, and AChE) and apoptotic body (histone H3) markers in EVs isolated by differential centrifugation (4000×g, 10,000×g, and 100,000×g) or filtration/ultracentrifugation (100,000×g) protocols. When we infected cells in the presence of caspase inhibitors, Hsc70 and AChE diminished in EVs obtained at 100,000×g, but not in EVs obtained at 4000×g or 10,000×g. In addition, caspase inhibitors decreased CD63 and AChE in vesicles with low and high buoyant densities. Without caspase inhibitors, RV infection increased exosome markers in all of the EVs obtained by differential centrifugation. However, CD63 preferentially localized in the 100,000×g fraction and H3 only increased in EVs concentrated at 100,000×g and with high buoyant densities on a sucrose gradient. Thus, RV infection increases the release of EVs that, upon concentration at 100,000×g, are composed by exosomes and apoptotic bodies, which can partially be separated using sucrose gradients.     

Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4⁺ and CD8⁺ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8⁺CM9⁺ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8⁺ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8⁺ T-cell responses.     

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg²⁺ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The ³H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs₂SO₄ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.     

Implications of fungal infections and mycotoxins in camel diseases in Saudi Arabia

Natural feed ingredients (corn, barley and wheat bran) and compound feed (manufactured pellet) are two types of fodder used for animal feeding, especially camel in Saudi Arabia. Twenty samples of each type of fodder were collected from seven different regions and screened for the presence of fungi, aflatoxins, ochratoxin and zearalenone. Fungal isolation of natural feed ingredients yielded 10 genera and 38 species of different fungi. Compound fodder samples were contaminated with 16 genera and 32 species of fungi. Total counts of Aspergillus, Penicillium and Fusarium in the animal feed samples were ranged from 54 to 223 × 10³, 31.9 to 60 × 10³ and 18 to 29 × 10³ CFU/g, respectively. These isolates when tested for aflatoxin, ochratoxin and zearalenone producing ability, revealed this property in only four isolate, identified as Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium graminaerum. The percentage of toxigenic fungi was ranged from 5.5% to 30% for natural feed ingredients and from 4.5% to 20% for compound feed. The incidence of aflatoxins (AFT) in samples of natural feed ingredients was found to be ranged from 1 to 24.8 ppb, ochratoxin A (OTA) ranged from 1 to 44 ppb and zearalenone (ZON) ranged from 1 to 23 ppb. Contamination of compound feed with aflatoxin and ochratoxin A was ranged from 1 to 6.4 ppb and 1 to 4.7 ppb, respectively. All samples collected were found contaminated with fungi or their toxins and natural feed samples were more contaminated compared to compound feed samples. The concentrations detected were in the allowed limit (<20 ppb) except four samples of natural feed ingredients which were above the allowed limit of the tested mycotoxins. In conclusion, feed samples were contaminated with fungi and some toxigenic isolates which were responsible about mycotoxin production. Some samples had exceeded amount of AFT, OTA and ZON and may be contaminated with other mycotoxins which mean implication of fungi in camel health problems and death in Saudi Arabia.     

Health Risk from the Use of Roof-Harvested Rainwater in Southeast Queensland,Australia, as Potable or Nonpotable Water,Determined Using Quantitative Microbial Risk Assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analyzed for the presence and numbers of zoonotic bacterial and protozoal pathogens using binary PCR and quantitative PCR (qPCR). Quantitative microbial risk assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from roof-harvested rainwater used as potable or nonpotable water. Of the 214 samples tested, 10.7%, 9.8%, 5.6%, and 0.4% were positive for the Salmonella invA, Giardia lamblia β-giardin, Legionella pneumophila mip, and Campylobacter jejuni mapA genes, respectively. Cryptosporidium parvum oocyst wall protein (COWP) could not be detected. The estimated numbers of Salmonella, G. lamblia, and L. pneumophila organisms ranged from 6.5 × 10¹ to 3.8 × 10² cells, 0.6 × 10° to 3.6 × 10° cysts, and 6.0 × 10¹ to 1.7 × 10² cells per 1,000 ml of water, respectively. Six risk scenarios were considered for exposure to Salmonella spp., G. lamblia, and L. pneumophila. For Salmonella spp. and G. lamblia, these scenarios were (i) liquid ingestion due to drinking of rainwater on a daily basis, (ii) accidental liquid ingestion due to hosing twice a week, (iii) aerosol ingestion due to showering on a daily basis, and (iv) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were (i) aerosol inhalation due to showering on a daily basis and (ii) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeded this threshold value and indicated that if undisinfected rainwater is ingested by drinking, then the incidences of the gastrointestinal diseases salmonellosis and giardiasis are expected to range from 9.8 × 10° to 5.4 × 10¹ (with a mean of 1.2 × 10¹ from Monte Carlo analysis) and from 1.0 × 10¹ to 6.5 × 10¹ cases (with a mean of 1.6 × 10¹ from Monte Carlo analysis) per 10,000 persons per year, respectively, in urban SEQ. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for use as potable water.Roof-harvested rainwater has received significant attention as a potential alternative source of potable and nonpotable water in regions where water is scarce (37). To encourage the use of roof-harvested rainwater, governmental bodies of many countries, such as Australia, Denmark, Germany, India, and New Zealand, are providing subsidies to residents to encourage the use of rainwater for domestic purposes. The use of rainwater is quite common in Australia, particularly in rural and remote areas, where reticulated mains or town water is not available. Recent water scarcity in several capital cities prompted the use of rainwater as an alternative source. For instance, the Queensland State Government initiated the “Home Water Wise Rebate Scheme,” which provides subsidies to Southeast Queensland (SEQ) residents who use rainwater as nonpotable water for domestic purposes (49). Over 260,000 householders were granted subsidies up to December 2008, when the scheme was concluded.There is a general community feeling that roof-harvested rainwater is safe to drink, and this is partially supported by limited epidemiological evidence (26). Some studies have reported that roof-harvested rainwater quality is generally acceptable for use as potable water (13, 29). In contrast, the presence of potential pathogens, such as Aeromonas spp. Campylobacter spp., Campylobacter jejuni, Salmonella spp., Legionella pneumophila, Giardia spp., Giardia lamblia, and Cryptosporidium spp., in roof-harvested rainwater samples has been reported (2, 9, 34, 45, 47, 48). Such pathogens can cause gastrointestinal illness in humans, with nausea, vomiting, and/or diarrhea occurring within 12 to 72 h (Salmonella enterica serovar Typhimurium) to 9 to 15 days (Giardia lamblia) after ingestion of contaminated water. L. pneumophila can cause the respiratory infection pneumonia, and the fatality rate can be 50% in immunocompromised patients (57).Direct routine monitoring of the microbiological quality of source water for all possible pathogens is not economically, technologically, or practically feasible. Consequently, traditional fecal indicators, such as fecal coliforms, Escherichia coli, and enterococci, have long been used to determine the presence of pathogens. Most studies assess the quality of roof-harvested rainwater based on the numbers of these fecal indicators (13, 30). However, the major limitation in using fecal bacteria as indicators is their poor correlation with the presence of pathogenic microorganisms in water (2, 30). An alternative is the measurement of pathogens using traditional culture-based methods. However, there are several limitations of such methods, including the underestimation of the bacterial number due to the presence of injured or stressed cells (10) and the fact that certain microorganisms in environmental waters can be viable but not culturable (39). Culture-based methods are also generally laborious and costly. Recent advances in molecular techniques such as PCR technology enable rapid, specific, and sensitive detection of many pathogens. Advances in PCR methodology also enable the quantification of potential pathogens in source water that are otherwise difficult and/or laborious to culture using traditional microbiological methods. In view of this, we used binary PCR (presence/absence)- and quantitative PCR (qPCR)-based assays to first detect and then quantify zoonotic pathogens in samples from roof-harvested rainwater in SEQ residential houses.The aims of the research study were 2-fold: (i) to quantify the number and frequency of occurrence of Salmonella, G. lamblia, and L. pneumophila organisms in a range of domestic water tanks in SEQ by using qPCR-based methods and (ii) to apply quantitative microbial risk assessment (QMRA) analysis in order to estimate the risk of infection from exposure to these pathogens found in roof-harvested rainwater. The uniqueness of this study stems from the fact that instead of measuring fecal indicators, the pathogens that are capable of causing illness were quantified and this information was combined with QMRA to assess the human health risk of using roof-harvested rainwater as potable or nonpotable water.