Investigating degradation metabolites and underlying pathway of azo dye "Reactive Black 5" in bioaugmented floating treatment wetlands.

The direct discharge of azo dyes and/or their metabolites into the environment may exert toxic, mutagenic, and carcinogenic effects on exposed fauna and flora. In this study, we analyzed the metabolites produced during the degradation of an azo dye namely Reactive Black 5 (RB5) in the bacterial-augmented floating treatment wetlands (FTWs), followed by the investigation of their underlying toxicity. To this end, a FTWs system was developed by using a common wetland plant Phragmites australis in the presence of three dye-degrading bacteria (Acinetobacter junii strain NT-15, Pseudomonas indoloxydans strain NT-38, and Rhodococcus sp. strain NT-39). We found that the FTW system effectively degraded RB5 into at least 20 different metabolites with the successful removal of color (95.5%) from the water. The fish toxicity assay revealed the nontoxic characteristics of the metabolites produced after dye degradation. Our study suggests that bacterially aided FTWs could be a suitable option for the successful degradation of azo dyes, and the results presented in this study may help improve the overall textile effluent cleanup processes.

Genotoxicity of aluminium oxide, iron oxide, and copper nanoparticles in mouse bone marrow cells.

The aim of this study was to evaluate the genotoxic effects of Al2O3, Fe2O3, and Cu nanoparticles with chromosomal aberration (CA), micronucleus (MN), and comet assays on the bone marrow of male BALB/c mice. Three doses of Al2O3, Fe2O3 (75, 150, and 300 mg/kg), or Cu (5, 10, and 15 mg/kg) nanoparticles were administered to mice through intraperitoneal injection once a day for 14 days and compared with negative control (distilled water) and positive control (mitomycin C and methyl methanesulphonate). Al2O3 and Fe2O3 did not show genotoxic effects, but Cu nanoparticles induced significant (P<0.05) genotoxicity at the highest concentration compared to negative control. Our findings add to the health risk information of Al2O3, Fe2O3, and Cu nanoparticles regarding human exposure (occupational and/or through consumer products or medical treatment), and may provide regulatory reference for safe use of these nanoparticles. However, before they can be used safely and released into the environment further chronic in vivo studies are essential.

Paper-based analytical devices for colorimetric detection of S. aureus and E. coli and their antibiotic resistant strains in milk.

Animal derived milk which is an important part of human diet due to its high nutritional value not only supports humans but also presents a growth environment for pathogenic bacteria. Milk may become contaminated with bacteria through udder infections or through contact within the dairy farm environment. Infections are treated with antibiotics, with ß-lactams most commonly used in veterinary medicine. However, their frequent use leads to the emergence of ß-lactam resistant bacterial strains, which causes difficulties in the treatment of infections in both humans and animals. Detection of pathogens as well as their antibiotic sensitivity is a pre-requisite for successful treatment and this is generally achieved with laboratory-based techniques such as growth inhibition assays, enzyme-linked immunosorbent assays (ELISA) or polymerase chain reactions (PCRs), which are unavailable in resource-limited settings. Here, we investigated paper-based analytical devices (µPADs) for the presumptive detection of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and their antibiotic resistant bacterial strains in milk samples. The µPADs were fabricated on filter paper using wax printing, and then impregnated with chromogenic substrates, which reacted with bacterial enzymes to form coloured products. Limits of detection of S. aureus and E. coli and their antibiotic resistant strains in milk samples were found to be 106 cfu mL-1. Enrichment of milk samples in a selective medium for 12 h enabled detection as low as 10 cfu mL-1. The paper devices tested on a set of 640 milk samples collected from dairy animals in Pakistan demonstrated more than 90% sensitivity and 100% selectivity compared to PCR, showing promise to provide inexpensive and portable diagnostic solutions for the detection of pathogenic bacteria in resource-limited settings.

Risk assessment of genetically modified sugarcane expressing AVP1 gene.

Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and policies for the regulations of genetically modified (GM) organisms. The potential health risks associated with GM sugarcane containing AVP1 gene confers resistance against drought and salinity were evaluated by animal feeding studies and some genotoxicity assays. Acute and sub-chronic toxicity examinations were carried out via oral dose administration of GM sugarcane juice supplemented with the normal diet (modified from certified rodent standard diet) on Wistar rats. AVP1 protein concentration in sugarcane juice was 1mg/1 mL. Biochemical, haematological blood analyses were performed and the results revealed that there were non-significant differences among all the treatment groups; GM sugarcane juice, non-GM sugarcane juice and the control group (normal diet and water). Genotoxicity assessment based on the comet assay and the micronucleus assay data exhibited that AVP1 GM sugarcane was not genotoxic or cytotoxic in rat's peripheral blood. These research findings supported the conclusion that GM AVP1 sugarcane was non-toxic in experimental animals. Therefore, data generated through this research work would be helpful for the commercial release of GM AVP1 sugarcane.

Genomic variants identified from whole-genome resequencing of indicine cattle breeds from Pakistan.

The primary goal of cattle genomics is the identification of genome-wide polymorphism associated with economically important traits. The bovine genome sequencing project was completed in 2009. Since then, using massively parallel sequencing technologies, a large number of Bos taurus cattle breeds have been resequenced and scanned for genome-wide polymorphisms. As a result, a substantial number of single nucleotide polymorphisms (SNPs) have been discovered across European Bos taurus genomes, whereas extremely less number of SNPs are cataloged for Bos indicus breeds. In this study, we performed whole-genome resequencing, reference-based mapping, functional annotation and gene enrichment analysis of 20 sires representing eleven important Bos indicus (indicine) breeds of Pakistan. The breeds sequenced here include: Sahiwal, Red Sindhi, Tharparkar and Cholistani (tropically adapted dairy and dual purpose breeds), Achai, Bhagnari, Dajal and Lohani (high altitude adapted dual and drought purpose breeds); Dhanni, Hisar Haryana and Gabrali (dairy and light drought purpose breeds). A total of 17.4 billion QC passed reads were produced using BGISEQ-500 next generation sequencing platform to generate 9 to 27-fold genome coverage (average ~16×) for each of the 20 sequenced sires. A total of 67,303,469 SNPs were identified, of which 3,850,365 were found novel and 1,083,842 insertions-deletions (InDels) were detected across the whole sequenced genomes (491,247 novel). Comparative analysis using coding region SNPs revealed a close relationship between the best milking indicine breeds; Red Sindhi and Sahiwal. On the other hand, Bhagnari and Tharparkar being popular for their adaptation to dry and extremely hot climates were found to share the highest number of SNPs. Functional annotation identified a total of 3,194 high-impact (disruptive) SNPs and 745 disruptive InDels (in 275 genes) that may possibly affect economically important dairy and beef traits. Functional enrichment analysis was performed and revealed that high or moderate impact variants in wingless-related integration site (Wnt) and vascular smooth muscle contraction (VSMC) signaling pathways were significantly over-represented in tropically adapted heat tolerant Pakistani-indicine breeds. On the other hand, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) signaling pathways were found over-represented in highland adapted Pakistani-indicine breeds. Similarly, the ECM-receptor interaction and Jak-STAT signaling pathway were significantly enriched in dairy and beef purpose Pakistani-indicine cattle breeds. The Toll-like receptor signaling pathway was significantly enriched in most of the Pakistani-indicine cattle. Therefore, this study provides baseline data for further research to investigate the molecular mechanisms of major traits and to develop potential genomic markers associated with economically important breeding traits, particularly in indicine cattle.

Characterization of Hydrocarbon-Degrading Bacteria in Constructed Wetland Microcosms Used to Treat Crude Oil Polluted Water.

Ten plant species were grown in constructed wetlands (CWs) to remediate water containing 2% (w/v) crude oil. The plant species with better growth and biomass production were Typha latifolia and Cyperus laevigatus, and they were significantly correlated (R2 = 0.91) with hydrocarbon degradation. From T. latifolia and C. laevigatus, 33 hydrocarbon-degrading bacterial strains were isolated from the rhizosphere, and root and shoot interiors. More diversified bacteria were found in the rhizosphere and endosphere of C. laevigatus than those of T. latifolia. The predominant cultural hydrocarbon-degrading bacteria were shown to belong to the genera Pseudomonas, Acinetobacter and Bacillus. In addition to genes involved in hydrocarbon degradation, most of the bacteria displayed multiple plant growth promoting (PGP) activities. This study suggests the importance of selecting suitable bacterial strains with hydrocarbon degradation and PGP activities for improving the efficacy of CWs used in remediating water contaminated with crude oil.

In silico identification of conserved miRNAs and their selective target gene prediction in indicine (Bos indicus) cattle.

The modern cattle was domesticated from aurochs, sharing its physiological traits into two subspecies Bos taurus and Bos indicus. MicroRNAs (miRNAs) are a class of non-coding short RNAs of ~22nt which have a key role in the regulation of many cellular and physiological processes in the animal. The current study was aimed to predict and annotate the potential mutations in indicine miRNAs throughout the genome using de novo and homology-based in silico approaches. Genome-wide mapping was performed in available indicine assembly by the homology-based approach and 768 miRNAs were recovered out of 808 reported taurine miRNAs belonging to 521 unique mature miRNA families. While 42 precursors were dropped due to lack of secondary miRNA structure, increasing stringency or decreasing similarity between the two genomes' miRNA. Increasing tendency of miRNAs incidence was observed on chr5, chr7, chr8, chr12 and chr21 with 19 polycistronic miRNA within 1-kilobase distance throughout the indicine genome. Notably, 12 miRNAs showed copy number variation. Eighteen miRNAs showed a mutation in their mature sequences in which eight were found in their seed region. Whilst in de novo based approach, 12 novel potential miRNAs on Y chromosome in indicine cattle along with a new miRNA (bind-miR-1264) on chrX were found. The final data set is annotated and explains the impending target genes that are responsible for enhanced immunity, heat tolerance and disease tolerance regulation in indicine. The study conforms to better understanding and perceptive approach towards indicine genome.

Genetic detection of peste des petits ruminants virus under field conditions: a step forward towards disease eradication.

BACKGROUND: The devastating viral disease of small ruminants namely Peste des petits ruminants (PPR) declared as target for "Global Eradication" in 2015 by the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (OIE). For a successful eradication campaign, molecular diagnostic tools are preferred for their specificity, efficacy and robustness to compliment prophylactic measures and surveillance methods. However, molecular tools have a few limitations including, costly equipment, multi-step template preparation protocols, target amplification and analysis that restrict their use to the sophisticated laboratory settings. As reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) has such an intrinsic potential for point of care diagnosis, this study focused on the genetic detection of causative PPR virus (PPRV) in field conditions. It involves the use of a sample buffer that can precipitate out virus envelope and capsid proteins through ammonium sulphate precipitation and exposes viral RNA, present in the clinical sample, to the LAMP reaction mixture. RESULTS: The test was evaluated using 11 PPRV cultures, and a total of 46 nasal swabs (n = 32 collected in the field outbreaks, n = 14 collected from experimentally inoculated animals). The RT-LAMP was compared with the reverse transcription-PCR (RT-PCR) and real-time quantitative RT-PCR (RT-qPCR) for its relative specificity, sensitivity and robustness. RT-LAMP detected PPRV in all PPRV cultures in or less than 30 min. Its detection limit was of 0.0001TCID50 (tissue culture infective dose-50) per ml with 10-fold higher sensitivity than that of RT-PCR. In 59.4% of the field samples, RT-LAMP detected PPRV within 35-55 min. The analytical sensitivity and specificity of the RT-LAMP were equivalent to that of the RT-qPCR. The time of detection of PPRV decreased by at least forty minutes or 3-4 h in case of in the RT-LAMP as compared with the RT-qPCR and the RT-PCR, respectively. CONCLUSIONS: The sensitive and specific RT-LAMP test developed in this study targeting a small fragment of the N gene of PPRV is a rapid, reliable and applicable molecular diagnostic test of choice under the field conditions. RT-LAMP requiring minimal training offers a very useful tool for PPR diagnosis especially during the "Global PPR Eradication Campaign".

Plant-bacteria partnerships for the remediation of persistent organic pollutants.

High toxicity, bioaccumulation factor and widespread dispersal of persistent organic pollutants (POPs) cause environmental and human health hazards. The combined use of plants and bacteria is a promising approach for the remediation of soil and water contaminated with POPs. Plants provide residency and nutrients to their associated rhizosphere and endophytic bacteria. In return, the bacteria support plant growth by the degradation and detoxification of POPs. Moreover, they improve plant growth and health due to their innate plant growth-promoting mechanisms. This review provides a critical view of factors that affect absorption and translocation of POPs in plants and the limitations that plant have to deal with during the remediation of POPs. Moreover, the synergistic effects of plant-bacteria interactions in the phytoremediation of organic pollutants with special reference to POPs are discussed.

In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types.

Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p < 0.01) increase in number of binucleated cells with micronuclei (96.6 ± 5.40) at the highest concentration (25 µg/mL). Copper NPs also induced DNA strand breaks at 10 µg/mL and oxidative DNA damage at 5 and 10 µg/mL. We consider these findings very useful in evaluating the genotoxic potential of NPs especially because of their increasing applications in human health and environment with limited knowledge of their toxicity and genotoxicity.

Enhanced degradation of textile effluent in constructed wetland system using Typha domingensis and textile effluent-degrading endophytic bacteria.

Textile effluent is one of the main contributors of water pollution and it adversely affects fauna and flora. Constructed wetland is a promising approach to remediate the industrial effluent. The detoxification of industrial effluent in a constructed wetland system may be enhanced by applying beneficial bacteria that are able to degrade contaminants present in industrial effluent. The aim of this study was to evaluate the influence of inoculation of textile effluent-degrading endophytic bacteria on the detoxification of textile effluent in a vertical flow constructed wetland reactor. A wetland plant, Typha domingensis, was vegetated in reactor and inoculated with two endophytic bacterial strains, Microbacterium arborescens TYSI04 and Bacillus pumilus PIRI30. These strains possessed textile effluent-degrading and plant growth-promoting activities. Results indicated that bacterial inoculation improved plant growth, textile effluent degradation and mutagenicity reduction and were correlated with the population of textile effluent-degrading bacteria in the rhizosphere and endosphere of T. domingensis. Bacterial inoculation enhanced textile effluent-degrading bacterial population in rhizosphere, root and shoot of T. domingensis. Significant reductions in COD (79%), BOD (77%) TDS (59%) and TSS (27%) were observed by the combined use of plants and bacteria within 72 h. The resultant effluent meets the wastewater discharge standards of Pakistan and can be discharged into the environment without any risks. This study revealed that the combined use of plant and endophytic bacteria is one of the approaches to enhance textile effluent degradation in a constructed wetland system.

Genotoxicity of chlorpyrifos in freshwater fish Labeo rohita using Alkaline Single-cell Gel Electrophoresis (Comet) assay.

Chlorpyrifos is a widely used insecticide of organophosphate group, which causes severe toxicological effects in non target aquatic organisms especially in fish. In the present study the genotoxic effects of sublethal concentrations of chlorpyrifos were observed in the erythrocytes and gill cells of Labeo rohita (commonly known as rohu) using the Alkaline Single-Cell Gel Electrophoresis (Comet) assay. Effects of chlorpyrifos on the behavior of the fish were also investigated. The 96 h LC50 value of chlorpyrifos, estimated by Trimmed Spearman-Karber (TSK) in static bioassay, was found to be 442.8 µg/L. On the basis of LC50 value, the fish were exposed to three sublethal concentrations of chlorpyrifos (SL-I ∼221.4 µg/L, SL- II ∼110.7 µg/L and SL-III ∼73.8 µg/L) for 96 h. Blood and gill samples were collected at every 24 h and were subjected to the Comet assay. The observed DNA damage was concentration dependent and time dependent and those levels of DNA damage in between the tested concentrations and times were significantly different (p < 0.01). It was also found that the gill cells are more sensitive to chlorpyrifos, though; it revealed more DNA damage as compared to the erythrocytes of fish. Fish exposed to different concentrations of chlorpyrifos showed different neurotoxic behavioral responses. It was concluded that chlorpyrifos is a genotoxic and neurotoxic insecticide causing DNA damage and neurotoxic effects in Labeo rohita.

RT-PCR evaluation for identification and sequence analysis of foot-and-mouth disease serotype O from 2006 to 2007 in Punjab, Pakistan.

FMD clinically positive 250 tissue samples (mouth and hoof epithelium and vesicle swabs, tongue tissue) and 175 secretion samples (milk, saliva, serum, plasma) were evaluated by RT-PCR for the diagnosis of FMD with different pair of universal and serotype-specific primers from 2006 to 2007 in Punjab, Pakistan. Universal primer pair P1/P2 from VP1 gene detected FMD in 182 out of 250 (72.8%) tissues and 92 out of 175 (52.6%) secretion samples, while universal primer 1F/1R from 5'UTR region detected FMD in 218 out of 250 (87.2%) tissues and 142 out of 175 (81.1%) secretion samples. 1F/1R proved better than the P1/P2 primer pair for primary diagnosis of FMD, direct from the clinical positive samples. Direct sequencing of the universal primer pair P1/P2 revealed that O serotype of FMD was circulating in this region. O serotype of FMD was detected with O-1C(ARS4)/PNK 61, AU(O)/AU(rev), AU(O)/PNK61 primer pairs, these primer pairs also compared with each other. AU(O)/AU(rev) and AU(O)/PNK61 detected O serotype of FMD in 88.9% tissue and swab (mouth and hoof vesicle swabs) samples and 71.9% different secretion (milk, saliva, serum, plasma) samples, while O-1C(ARS4)/PNK 61 detected 48.1% tissue and swab (mouth and hoof vesicle swabs) samples and 37.5% different secretion (milk, saliva, serum, plasma) samples. AU(O)/AU(rev), AU(O)/PNK61 primer pairs detected 40.8% more tissue and swab samples, while these pairs detected 34.4% more secretion samples. Cloning of PCR product of AU(O)/AU(rev) VP1 gene and sequencing for phylogenetic studies revealed that O serotype of FMD circulating in Punjab, Pakistan was genetically very diverse from the 'O' serotype in Middle East and Europe. The dendrogram showed that Pakistan 'O' serotype was very much similar genetically to its neighbor countries (Sri Lanka, India, Iran, Iraq, and China) and PanAsia 1 lineage which caused 2001-outbreak in UK and 1994-outbreak in Saudi Arabia, etc.

Evaluation of the acute toxicity of profenofos and its effects on the behavioral pattern of fingerling common carp (Cyprinus carpio L., 1758).

Profenofos, an organophosphate insecticide is acetylcholinesterase inhibitor that has the potential to contaminate the ground water. The 96 h LC(50) value of profenofos was determined in 3-month-old fingerling common carp (Cyprinus carpio) with a body weight 1.04 +/- 0.25 g and a body length 4.25 +/- 0.75 cm at 26 +/- 1 degrees C temperature. Trimmed Spearman-Karber (TSK) software was used for the statistical analysis, which calculated the LC(50) value as 62.4 microg/L for three replicates of the assay. The behavioral responses of fish exposed to profenofos included loss of balance, moving in spiral fashion with sudden jerky movements, lying on their sides and rapid flapping of the operculum with the mouth open.