Long-term monitoring of the biomass and production of lipids by Nitzschia palea for biodiesel production.

Pennate diatom Nitzschia palea can be cultured in outdoor vertical-bed photobioreactors to produce biodiesel. To assess the production of biomass and lipids, non-axenic cultures of Nitzschia palea were grown outdoors, and the growth of these cultures was measured biweekly. During the annual cycle of algal culture, the culture temperature ranged from 17.3 °C to 33.5 °C, the dry weight biomass ranged from 0.11 g l-1 to 0.25 g l-1, light energy] ranged from 1.94 Wm-2 to 3.9 Wm-2 and intracellular lipid content ranged from 7.1% to 11.4% of biomass weight after drying at 60 °C. Gas chromatography/mass spectroscopy (GC/MS) analysis of n-hexane extracts showed that the intracellular lipids were primarily C14:0 myristic acid (9.01%), C15:0 pentadecyclic acid (8.26%) and two types of C16:0, palmitic acid (41.13%) and palmitoleic acid (29.25%). Gel permeation analysis showed that carboxylic acids comprised 28.9% of lipids, 16.3% of monoglycerides, 27.3% of diglycerides and 24.3% of triglycerides. Alcoholysis of lipids resulted in the conversion of about 93.9% of fatty acids to equivalent fatty acid methyl esters (FAME) or biodiesel, which, on basis of wt%, consisted primarily of C15:0 methyl myristate (8.3%), C16:0 methyl pentadecanoate] (7.2%), C17:1methyl palmitoleate (28.7%) and methyl palimtate](39.8%).

Propidium monoazide conventional PCR and DNA sequencing: detection of negative culture bacterial pathogens causing subclinical mastitis.

AIMS: This study was conducted to early detect the negative culture bacterial pathogens causing subclinical mastitis for the fast diagnosis of the disease and the reduction of some milk-transmitted pathogenic bacteria to human consumers. METHODS AND RESULTS: A total of 171 positive California mastitis test (CMT) milk samples collected from asymptomatic dairy cows in Sharkia Governorate, Egypt were examined by conventional bacteriological methods. The obtained results revealed that Streptococcus species (77·2%), followed by Staphylococcus species (48·6%) and Escherichia coli (25·7%) were the most predominant bacterial pathogens isolated from positive culture milk samples, whereas Enterobacter and Pseudomonas species were the lowest ones (1·2%, for each). Herein, 13 (7.6%) negative culture milk samples were subjected to propidium monoazide (PMA) conventional PCR assay, followed by DNA sequencing of purified PCR amplicons. Sequence analysis identified seven different types of negative culture bacterial pathogens comprising as following; 4 Enterococcus hirae, 2 Bacillus cereus, 2 Staphylococcus aureus, 1 Bacillus mycoides, 1 Bacillus subtilis, 1 Enterococcus faecium and 1 Escherichia coli. CONCLUSIONS: All the detected negative culture bacterial pathogens by PMA-PCR assay, followed by DNA sequencing were incriminated in causing subclinical mastitis disease and had serious implications on human public health through consumption of milk contaminated with those recovered bacterial pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The used methods could be useful in the routine detection of negative culture bacterial pathogens present in milk and consequently, it will help in the rapid diagnosis of subclinical mastitis disease and the reduction of many milk-transmitted diseases to human.

Impact of single and mixed infections with Escherichia coli and Mycoplasma gallisepticum on Newcastle disease virus vaccine performance in broiler chickens: an in vivo perspective.

AIMS: This study was designed to investigate, in an in vivo setting, the effects of single and combined infections with either Mycoplasma gallisepticum (MG) and/or Escherichia coli on the chicken immune response induced by Newcastle disease virus (NDV) vaccine. METHODS AND RESULTS: Humoral immunity was measured through detection of NDV antibody and anti-NDV IgG titres using haemagglutination-inhibition test and enzyme-linked immunosorbent assay, respectively. In addition, the expression levels of pro-inflammatory cytokines' genes (interleukin (IL) 6, IL4 and interferon (IFN) γ) were analysed using quantitative reverse transcription PCR. Significant (P < 0·05) results in all immunological parameters were detected in the vaccinated noninfected chicken group in comparison with those in groups exposed to bacterial infections. Bacterial infection along with vaccination hampered the NDV antibodies production and reduced the vaccine upregulated cytokine genes. The vaccinated mixed infection group reported lower antibody titres and cytokines expression levels compared to those in the single infection groups. All the previously enhanced immunological parameters reflected the maximum protection post challenge with velogenic viscerotropic NDV in the vaccinated noninfected chicken group. CONCLUSIONS: These findings provide novel insights into the immunosuppression activities of MG and E. coli infection in chickens vaccinated against NDV. SIGNIFICANCE AND IMPACT OF THE STUDY: This study hopes to provide a better insight to the immunosuppressive action of bacterial pathogens in chickens. This will help to improve biosecurity strategies during NDV vaccination in the future.

What is behind phylogenetic analysis of hospital-, community- and livestock-associated methicillin-resistant Staphylococcus aureus?

Methicillin-resistant Staphylococcus aureus (MRSA) has been shown to be the predominant life-threatening pathogen in Egypt. MRSA is a major cause of severe healthcare-associated (HA) infections. During the last decades, the incidence of community-associated (CA) MRSA infections has a complex epidemiology arising from the circulation of different strains in the general population. Moreover, livestock-associated (LA) MRSA emerged recently becomes an emerging threat to public health. Therefore, it is important to illuminate the differences between CA-, HA- and LA-MRSA to shed light on their genetic diversity and evolution. This study presents the first data on analysing the correlation between CA-, LA- and HA-MRSA using antibiogram typing, molecular characteristics and antimicrobial resistance and virulence genes' profiles. Overall, HA-MRSA strains tended to be multidrug resistant and less virulent than both LA- and CA-MRSA strains. Importantly, CA-MRSA strains had a high homology with each of HA- and LA-MRSA. However, no similarity was observed between HA- and LA-MRSA. Our findings suggest that the epidemiological changes in genetic behaviour between HA- and LA-MRSA are due to the presence of CA-MRSA confirming that CA-MRSA has created a public health crisis worldwide.

Genetic basis of resistance waves among methicillin resistant Staphylococcus aureus isolates recovered from milk and meat products in Egypt.

Antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem for clinicians worldwide. The present study attempted to evaluate the susceptibility patterns of MRSA to various antimicrobials and the prevalence of inducible clindamycin resistance as well as the relevant antibiotic and antiseptic resistance genes among these isolates. Totally, 40 MRSA isolates were recovered from examined milk and meat product samples (18.60%). Multi-drug resistance (MDR) was remarkably observed among 85% of these isolates. There was a good correlation between phenotypic determination of methicillin, amoxicillin/clavulinic acid and tetracycline resistances and PCR detections of mecA, blaZ and tet(K) genes, respectively, but norA gene was not detected in the four ciprofloxacin resistant isolates. Although, 55% of MRSA expressed resistance to benzalkonium chloride (BC), neither qacA/B nor smr gene was detected. Of 20 isolates exhibiting erythromycin- clindamycin discordant resistance pattern, 8 displayed positive double disk diffusion (D-zone) test denoting inducible macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype with the inducibly expressed erm(A) and erm(C) genes in 87.5% of these isolates. Besides, the remaining 12 isolates showed MS phenotype (resistant to macrolides and type B streptogramins only) with a variety of erm(A), mph(C), msr(A) or a combination of these genes including erm(C). Finally, the constitutive MLSB phenotype with the constitutive expression of erm(A), erm(B) and erm(C) genes was comprised in 2 isolates with higher minimum inhibitory concentration (MIC) values for erythromycin (512 and 1024 µg/ml) and clindamycin (16 and 32 µg/ml). These findings suggested the importance of monitoring the evolution of MRSA resistance.

Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems.

Trichothecium roseum Causes Fruit Rot of Tomato, Orange, and Apple in Pakistan.

During a field survey of greenhouses and fresh markets in 2013, fruits of tomato, oranges, and apples exhibited rot symptoms with white mycelial growth and salmon-color sporulation in the vicinity of Sargodha city (32°5'1″ N, 72°40'16″ E), Pakistan. Diseased fruit samples were collected in plastic bags and taken to laboratory on ice for further diagnosis. Diseased fruits were observed under a stereo microscope and single spores were removed using an inoculating needle. Isolation from single spores showed pink to white colonies on potato dextrose agar (PDA) containing hyaline, 2-celled, ellipsoid to pyriform conidia (17 to 24 × 7 to 11 µm) with slanting and truncate basal mark and produced in clusters. Conidiophores were branched (105 to 254 × 2 to 4 µm) and hyphae were hyaline (3 to 5 µm in diameter). These characteristics of the fungus were similar to Trichothecium roseum (Pers.) as reported by Inácio et al. (1). Genomic DNA was extracted by using CTAB buffer from a single pure colony of one isolate of the fungus and PCR analysis was performed for ITS region and part of the 5' end of the beta tubulin (TUB) gene (2,3). Single fragments of 550 bp and 1.5 kb length from ITS and TUB gene were amplified and sequenced (GenBank Accession Nos. KF975702 and KJ607590, respectively). Sequence analysis showed 99% similarity with T. roseum isolates from different regions of the world. Phylogenetic analysis (MEGA version 5.2 with WAG model) showed the close relatedness to the isolates of T. roseum from Pakistan and isolates from other parts of the world that revealed the low genetic variability of ITS region. TUB gene sequence analysis indicated 100% homology with isolates of T. roseum and to the other species in Hypocreales. Pathogenicity tests were performed on tomato cvs. Nova Mech and Rio Grande, orange cv. Kinnow, and on apple cv. Golden Delicious by inoculating five fruits from each cultivar. Spore suspensions (105 conidia/ml of sterilized distilled water) were inoculated into all wounded fruits (9 wounds/fruit) of each cultivar and incubated at 25°C for the development of symptoms. Five wounded fruits of each cultivar were inoculated with sterilized distilled water as a control treatment. The fruits were kept in plastic boxes and incubated in humid chambers for 5 days. The symptoms on apples were observed as brown rot with pinkish spores on rotted tissue. The cross section of apple fruits also showed the brown rotted tissues internally. The fungus developed mycelium and spores on the surface and caused severe rotting inside the tomato and citrus fruits. T. roseum was re-isolated by picking a single spore from rotted tissues of fruits under a stereo microscope, and culturing on PDA. The re-isolated fungus was confirmed morphologically and by molecular techniques. Tomato and apple has been reported as a host for T. roseum (1,4,5) but oranges have not. To our knowledge, this is the first record of T. roseum infecting tomato, oranges, and apples in Pakistan. References: (1) C. A. Inácio et al. Plant Dis. 95:1318. 2011. (2) K. O'Donnell, and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (4) Y. H. Yun et al. Afr. J. Microbiol. Res. 7:1128, 2013. (5) M. Zabka et al. Mycopathologia. 162:65, 2006.

Role of ginger against the reproductive toxicity of aluminium chloride in albino male rats.

The aim of the current study was to evaluate reproductive disorders concomitant with aluminium chloride (AlCl(3) ) toxicity in Albino male rats. Attention was also directed to study the protective influence of ginger against this toxicity. Forty-five mature Albino Wistar male rats were equally divided into three groups; the first group was served as control group while those of the second group (AlCl(3) ) were daily treated with 34 mg/kg bw. AlCl(3) orally. The third group (AlCl(3) + ginger) was treated daily with AlCl(3) as in group 2 in combination with ginger (40 mg/kg bw), which started 2 weeks prior to AlCl(3) . Five animals from each group were sacrificed on days 30, 45 and 60 of treatment. AlCl(3) administration significantly decreased serum testosterone levels, increased testicular homogenate malondialdehyde and deteriorated semen picture with increased testicular DNA fragmentation. Histopathological examination revealed degenerative changes of the seminiferous tubules with focal areas of necrosed spermatogenic cells, marked degeneration and desquamation of the lining epithelial cells of epididymis as well as multiple calcified material in prostate gland following 60 days of aluminium treatment. Ginger treatment started to improve significantly all studied parameters after 60 days as compared with AlCl(3) -treated group. In the current study, it was concluded that AlCl(3) had a destructive effect on all the studied reproductive parameters. Treatment with ginger has an ameliorating effect against AlCl(3) toxicity after 60 days post-treatment.

Adriblastina-single stranded DNA interaction with statistical analysis.

The electrochemical behavior of adriblastina has been studied at in situ mercury film electrode (in situ MFE) and platinum electrode (PtE) in the presence of phosphoric acid as supporting electrolyte using Osteryoung square-wave stripping voltammetry (OSWSV) and cyclic voltammetry (CV). Optimal experimental and operational parameters have been selected for the drug accumulation and determination in aqueous medium. The interaction of the drug with single stranded DNA (ssDNA) has been studied and validated by using classical least square and partial least square with propagation of error. The formal potentials E degrees and E degrees ' and the equilibrium constants K(1) and K(2) have been calculated. It was found that K(2) for the oxidized form of adriblastina is 63 times than K(1) for the reduced form. Among several possible interfering metal ions, a complex formation reaction was observed between adriblastina and Cu(II) ions at in situ MFE. Cu(II) ions formed 1:2 metal:drug complex which is more stable than ssDNA-drug interaction and consequently it inhibits drug biochemical damage effects. The copper complex offers sub-nanograms determination of adriblastina in that 5.80 and 180pg/ml could be easily detected in aqueous and urine media, respectively, with R.S.D. less than 4%. F-test and t-test have been applied in urine media giving good results that indicated the high accuracy of the proposed method.

Osteryoung square wave stripping voltammetry at mercury film electrode for monitoring ultra trace levels of Tarabine PFS and its interaction with ssDNA.

The electrochemical oxidation and reduction behaviour of adsorbed species of antimetabolic antineoplastic agent Tarabine PFS (Cytosar-U) in Sorensen buffer solution of different pH values at an in situ-mercury film electrode (MFE) is studied using cyclic voltammetry (CV) and Osteryoung square-wave stripping voltammetry (OSWSV). Optimal experimental and operational parameters have been selected for the drug preconcentration and determination in aqueous medium. Based on the adsorption and accumulation of Tarabine PFS using Osteryoung square-wave anodic stripping voltammetry (OSWASV) at MFE, the drug is easily detected as 0.134 ng/ml (5.51 x 10(-10) M). Calibration plots have been constructed at different accumulation times. The standard deviation (n=10) at a concentration level of 6 x 10(-8) M Tarabine PFS is 0.062. The interaction of ssDNA with the drug under the optimal conditions at pH 7.7 has been studied. The formal potentials E degrees and E degrees ' and the equilibrium constants K(1) and K(2) have been calculated for the free form of Tarabine PFS and the bonded form with ssDNA, respectively. It was found that K(2) value for the bonded oxidized form is 298 times than that of K(1) for the bonded reduced form. Therefore, ssDNA has been found to interact strongly with the oxidized form of the drug. The method has been used for the nanogram determination of ssDNA with 1.9% variation coefficient. Detection limit of 3 ng/ml ssDNA has been achieved. Possible interfering organic compounds, cations and anions have been tested. The method has been applied for the drug determination in urine samples, down to 0.23 ng/ml could be easily achieved in such samples.

Studies on the phytoplankton populations and physico-chemical conditions of treated sewage discharged into Lake Manzala in Egypt.

Over a full year, the phytoplankton populations and physico-chemical conditions of treated sewage discharged into Lake Manzala in Egypt were investigated. Sixty-seven species of algae were identified, 18 Cyanophyta (Cyanobacteria), 19 Chlorophyta, 21 Bacillariophyta, 6 Euglenophyta, 2 Cryptophyta and one species Pyrrhophyta. Nitzschia (6 spp.), Scenedesmus (6 spp.), Navicula (4 spp.), Oscillatoria (4 spp.) and Euglena (4 spp.) were the most common genera. A remarkable seasonal variation in species composition and standing crop of the phytoplankton populations was noted during the study. The total phytoplankton standing crop appeared to be mainly dependent on the growth of certain species viz., Oscillatoria chalybea, O. princepes, O. tenuis, Microcystis aeruginosa, Anabaena constricta (Cyanophyta), Nitzschia obtusa, Bacillaria paradoxa, Cocconeis placentula, Cyclotella meneghiniana (Bacillariophyta), Pandorina morum, Volvox sp. (Chlorophyta) and Phacus curvicauda (Euglenophyta). The continuous presence of Anabaena constricta and Nitzschia palea was recorded in the treated sewage. The least represented algal divisions were Pyrrhophyta and Cryptophyta, both in terms of quality and quantity. The data indicate that the secondary effluents were unstable in their chemical features and grossly polluted. Therefore, the treatment systems must treat the discharged sewage to a tertiary level before discharging into Lake Manzala.

Effect of treated sewage on the water quality and phytoplankton populations of Lake Manzala (Egypt) with emphasis on biological assessment of water quality.

The effect of treated sewage on the quality of water and phytoplankton populations of Lake Manzala was studied with emphasis on use of algae to monitor water pollution as part of a search for a biological assessment of water quality. Lake Manzala is situated at the northern part of the Nile-delta, Egypt. Disposal of treated sewage into Lake Manzala appeared to have differential effects on water quality and phytoplankton populations. Marked seasonal and local variations were observed for the physical and chemical characteristics of water. 157 species of algae were identified, 59 Chlorophyta, 37 Bacillariophyta, 30 Cyanophyta (Cyanobacteria), 28 Euglenophyta, one Pyrrhophyta and 2 Cryptophyta. Distribution and abundance of these algal divisions were found to differ at different sampling stations. Qualitative and quantitative growth of each algal division displayed great seasonal variations. The phytoplankton standing crop was mainly due to the contribution of Bacillariophyta whereas the species composition is dependent mainly on Chlorophyta. A great parallelism was noted between the quality of water samples based upon the chemical and physical investigations and their quality based upon the biological indices. Compound eutrophication index indicated that the nature of the investigated water ranged between eutrophic and hypereutrophic conditions. Diversity index values indicated that the water in the study area was of a moderate level of pollution. Saprobic index and saprobic quotient revealed the presence of beta- to alpha-mesosaprobic forms of algae.