Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

An Inquiry of Changes in Environmental Governance by Community-driven Process

Urban environmental depletion has been a critical problem among industrialized-transformed societies, especially at the local level where administrative authorities’ capacity lags behind changes. Derived from governance concept, the idea of civil society inclusion is highlighted. Focusing on an agglomerated case study, Bang Plee Community in Thailand, this research investigates on a non-state sector, 201-Community organization, as an agent for changes to improve urban environments on solid waste collection. Two roles are contested: as an agent for neighborhood internal change and as an intermediary toward governance changes in state-civil society interaction. By employing longitudinal analysis via a project intervention as research experiment, the outcomes of both roles are detected portrayed in three spheres: state, state-civil society interaction, and civil society sphere. It discovers in the research regarding agglomerated context that as an internal changes for environmental betterment, 201-Community organization operation brings on waste reduction at the minimal level. Community-based organization as an agent for changes – despite capacity input it still limited in efficiency and effectiveness – can mobilize fruitfully only at the individual and network level of civil society sectors, while fails managing at the organizational level. The positive outcomes result by economic waste incentive associated with a limited-bonded group rather than the rise of awareness at large. As an intermediary agent for shared governance, the community-based organization cannot bring on mutual dialogue with state as much as cannot change the state’s operation arena of solid waste management. The findings confine the shared governance concept that it does not applicable in agglomerated locality as an effective outcome, both in terms of being instrumental toward civil society inclusion and being provocative of internal change. Shared environmental governance as summarized in this research can last merely a community development action. It distances significantly from civil society inclusion and empowerment. However, the research proposes that community-based environmental management and shared governance toward civil society inclusion in urban environmental improvement are still an expectable option and reachable if their factors and conditions of key success and failure are intersected with a particular context. Further studies demand more precise on scale, scope, and theses factors of environmental management operation operated by civil society sectors.

Nutrient cycling and nutrient use efficiency in urban and peri-urban agriculture of Kabul, Afghanistan

Like elsewhere also in Kabul, Afghanistan urban and peri-urban agriculture (UPA) has often been accused of being resource inefficient and unsustainable causing negatives externalities to community health and to the surroundings. These arise from the inappropriate management and use of agricultural inputs, including often pesticides and inter-city wastes containing heavy metal residues and pathogens. To address these concerns, parallel studies with the aims of quantification of carbon (C), nitrogen (N), phosphorus (P) and potassium (K) horizontal and vertical fluxes; the assessment of heavy metal and pathogen contaminations of UPA produce, and an economic analysis of cereal, vegetable and grape production systems conducted for two years in UPA of Kabul from April 2008 to October 2009. The results of the studies from these three UPA diverse production systems can be abridged as follows: Biennial net balances in vegetable production systems were positive for N (80 kg ha-1 ), P (75 kg ha-1) and C (3,927 kg ha-1), negative for K (-205 kg ha-1), whereas in cereal production systems biennial horizontal balances were positive for P (20 kg ha-1 ) and C (4,900 kg ha-1) negative for N (-155 kg ha-1) and K (-355 kg ha-1) and in vineyards corresponding values were highly positive for N (295 kg ha-1), P (235 kg ha-1), C (3,362 kg ha-1) and slightly positive for K (5 kg ha-1). Regardless of N and C gaseous emissions, yearly leaching losses of N and P in selected vegetable gardens varied from 70 - 205 kg N ha-1 and 5 - 10 kg P ha-1. Manure and irrigation water contributed on average 12 - 79% to total Inputs of N, P, K and C, 10 - 53% to total inputs of C in the gardens and fields. The elevated levels of heavy metal and pathogen loads on fresh UPA vegetables reflected contamination from increasing traffic in the city, deposits of the past decades of war, lacking collection and treatment of raw inter-city wastes which call for solutions to protect consumer and producer health and increase reliability of UPA productions. A cost-revenue analysis of all inputs and outputs of cereal, vegetable and grapes production systems over two years showed substantial differences in net UPA household income. To confirm these results, more detailed studies are needed, but tailoring and managing the optimal application of inputs to crop needs will significantly enhance farmer’s better revenues as will as environmental and produce quality.

Transaction costs of farmers’ participation in forest management: Policy implications of payments for environmental services schemes in Vietnam

Recent research on payments for environmental services (PES) has observed that high transaction costs (TCs) are incurred through the implementation of PES schemes and farmer participation. TCs incurred by households are considered to be an obstacle to the participation in and efficiency of PES policies. This study aims to understand transactions related to previous forest plantation programmes and to estimate the actual TCs incurred by farmers who participated in these programmes in a mountainous area of northwestern Vietnam. In addition, this study examines determinants of households’ TCs to test the hypothesis of whether the amount of TCs varies according to household characteristics. Results show that average TCs are not likely to be a constraint for participation since they are about 200,000 VND (USD 10) per household per contract, which is equivalent to one person’s average earnings for about two days of labour. However, TCs amount to more than one-third of the programmes’ benefits, which is relatively high compared to PES programmes in developed countries. This implies that rather than aiming to reduce TCs, an appropriate agenda for policy improvement is to balance the level of TCs with PES programme benefits to enhance the overall attractiveness of afforestation programmes for smallholder farmers. Regression analysis reveals that education, gender and perception towards PES programmes have significant effects on the magnitude of TCs. The analyses also points out the importance of local conditions on the level of TCs, with some unexpected results.

Application of a double bootstrap to investigation of determinants of technical efficiency of farms in Central Europe

The paper provides one of the first applications of the double bootstrap procedure (Simar and Wilson 2007) in a two-stage estimation of the effect of environmental variables on non-parametric estimates of technical efficiency. This procedure enables consistent inference within models explaining efficiency scores, while simultaneously producing standard errors and confidence intervals for these efficiency scores. The application is to 88 livestock and 256 crop farms in the Czech Republic, split into individual and corporate.

Application of a double bootstrap to investigation of determinants of technical efficiency of farms in Central Europe

The paper provides one of the first applications of the double bootstrap procedure (Simar and Wilson 2007) in a two-stage estimation of the effect of environmental variables on non-parametric estimates of technical efficiency. This procedure enables consistent inference within models explaining efficiency scores, while simultaneously producing standard errors and confidence intervals for these efficiency scores. The application is to 88 livestock and 256 crop farms in the Czech Republic, split into individual and corporate.

The use of life-cycle assessment to evaluate the environmental impacts of growing genetically modified, nitrogen use-efficient canola

Agriculture, particularly intensive crop production, makes a significant contribution to environmental pollution. A variety of canola (Brassica napus) has been genetically modified to enhance nitrogen use efficiency, effectively reducing the amount of fertilizer required for crop production. A partial life-cycle assessment adapted to crop production was used to assess the potential environmental impacts of growing genetically modified, nitrogen use-efficient (GMNUE) canola in North Dakota and Minnesota compared with a conventionally bred control variety. The analysis took into account the entire production system used to produce 1 tonne of canola. This comprised raw material extraction, processing and transportation, as well as all agricultural field operations. All emissions associated with the production of 1 tonne of canola were listed, aggregated and weighted in order to calculate the level of environmental impact. The findings show that there are a range of potential environmental benefits associated with growing GMNUE canola. These include reduced impacts on global warming, freshwater ecotoxicity, eutrophication and acidification. Given the large areas of canola grown in North America and, in particular, Canada, as well as the wide acceptance of genetically modified varieties in this area, there is the potential for GMNUE canola to reduce pollution from agriculture, with the largest reductions predicted to be in greenhouse gases and diffuse water pollution.

Improving the efficiency of energy utilization in cattle

The efficiency of energy utilisation in cattle is a determinant of the profitability of milk and beef production, as well as their environmental impact. At an animal level, meat and milk production by ruminants is less efficient than pig and poultry production, in part due to lower digestibility of forages compared with grains. However, when compared on the basis of human-edible inputs, the ruminant has a clear efficiency advantage. There has been recent interest in feed conversion efficiency (FCE) in dairy cattle and residual feed intake, an indicator of FCE, in beef cattle. Variation between animals in FCE may have genetic components, allowing selection for animals with greater efficiency and reduced environmental impact. A major source of variation in FCE is feed digestibility, and thus approaches that improve digestibility should improve FCE if rumen function is not disrupted. Methane represents a substantial loss of digestible energy from rations. Major determinants of methane emission are the amount of feed consumed and the proportions of forage and concentrates fed. In addition, feeding fat has long been known to reduce methane emission. A myriad of other supplements and additives are currently being investigated as mitigators of methane emission, but in many cases compounds effective in sheep are ineffective in lactating dairy cows. Ultimately, the adoption of ‘best practice’ in diet formulation and management may be the most effective option for reducing methane. In assessing the efficiency of energy use for milk and meat production by cattle, and their environmental impact, it is imperative that comparisons be made at a systems level, and that the wider social and economic implications of mitigation policy are considered.

Provision of environmental output within a multi-output distance function approach

This paper redefines technical efficiency by incorporating provision of environmental goods as one of the outputs of the farm. The proportion of permanent and rough grassland to total agricultural land area is used as a proxy for the provision of environmental goods. Stochastic frontier analysis was conducted using a Bayesian procedure. The methodology is applied to panel data on 215 dairy farms in England and Wales. Results show that farm efficiency rankings change when provision of environmental outputs by farms is incorporated in the efficiency analysis, which may have important political implications.

Building energy efficiency for sustainable development in China: challenges and opportunities

Rapid urbanisation in China has resulted in great demands for energy, resources and pressure on the environment. The progress in China's development is considered in the context of energy efficiency in the built environment, including policy, technology and implementation. The key research challenges and opportunities are identified for delivering a low carbon built environment. The barriers include the existing traditional sequential design process, the lack of integrated approaches, and insufficient socio-technical knowledge. A proposed conceptual systemic model of an integrated approach identifies research opportunities. The organisation of research activities should be initiated, operated, and managed in a collaborative way among policy makers, professionals, researchers and stakeholders. More emphasis is needed on integrating social, economic and environmental impacts in the short, medium and long terms. An ideal opportunity exists for China to develop its own expertise, not merely in a technical sense but in terms of vision and intellectual leadership in order to flourish in global collaborations.

Hierarchical analysis of production efficiency in a coastal trawl fishery

We present, pedagogically, the Bayesian approach to composed error models under alternative, hierarchical characterizations; demonstrate, briefly, the Bayesian approach to model comparison using recent advances in Markov Chain Monte Carlo (MCMC) methods; and illustrate, empirically, the value of these techniques to natural resource economics and coastal fisheries management, in particular. The Bayesian approach to fisheries efficiency analysis is interesting for at least three reasons. First, it is a robust and highly flexible alternative to commonly applied, frequentist procedures, which dominate the literature. Second,the Bayesian approach is extremely simple to implement, requiring only a modest addition to most natural-resource economist tool-kits. Third, despite its attractions, applications of Bayesian methodology in coastal fisheries management are few.

Social and environmental report assurance: some interview evidence

The steady growth of social and environmental reporting (SER) is being accompanied by an increase in social and environmental reporting assurance (SERA). The existing literature on SERA suggests that it is necessary to build credibility and trust among corporate stakeholders. Prior work has also found evidence of managerial and professional capture of SERA. In this paper, we present empirical evidence from interviews with corporate social responsibility representatives from 20 UK listed companies on whether they consider SERA to be necessary. We believe this to be the first research into SERA that uses an interview method. Our interviews revealed mixed feelings. Half of the respondents believed that external SERA would enhance credibility and trust which confirmed the prior literature. However, the other half believed that external SERA was not necessary, believing that internal assurance was sufficient. This was because they saw SERA as predominantly a managerial tool, useful for checking the efficiency of internal management control systems, rather than as a mechanism for enhancing corporate accountability to stakeholders and building credibility and trust. The potential for SERA to be a mechanism whereby greater dialogue is created between companies and their stakeholders on social and environmental issues is not being harnessed. This paper thus demonstrates a fundamental difference between the external prior normative literature and the managerial motivation in the SERA area.

Influence of earthworm Eisenia fetida on removal efficiency of N and P in vertical flow constructed wetland.

This study investigates biomass, density, photosynthetic activity, and accumulation of nitrogen (N) and phosphorus (P) in three wetland plants (Canna indica, Typha augustifolia, and Phragmites austrail) in response to the introduction of the earthworm Eisenia fetida into a constructed wetland. The removal efficiency of N and P in constructed wetlands were also investigated. Results showed that the photosynthetic rate (P n), transpiration rate (T r), and stomatal conductance (S cond) of C. indica and P. austrail were (p < 0.05) significantly higher when earthworms were present. The addition of E. fetida increased the N uptake value by above-ground of C. indica, T. augustifolia, and P. australis by 185, 216, and 108 %, respectively; and its P uptake value increased by 300, 355, and 211 %, respectively. Earthworms could enhance photosynthetic activity, density, and biomass of wetland plants in constructed wetland, resulting in the higher N and P uptake. The addition of E. fetida into constructed wetland increased the removal efficiency of TN and TP by 10 and 7 %, respectively. The addition of earthworms into vertical flow constructed wetland increased the removal efficiency of TN and TP, which was related to higher photosynthetic activity and N and P uptake. The addition of earthworms into vertical flow constructed wetland and plant harvests could be the significantly sustainable N and P removal strategy

Improving EU biofuels policy? Greenhouse gas emissions, policy efficiency, and WTO compatibility

Both the EU’s Renewable Energy Directive (RED) and Article 7a of its Fuel Quality Directive (FQD) seek to reduce greenhouse gas (GHG) emissions from transport fuels. The RED mandates a 10% share of renewable energy in transport fuels by 2020, whilst the FQD requires a 6% reduction in GHG emissions (from a 2010 base) by the same date. In practice, it will mainly be biofuels that economic operators will use to meet these requirements, but the different approaches can lead to either the RED, or the FQD, acting as the binding constraint. A common set of environmental sustainability criteria apply to biofuels under both the RED and the FQD. In particular, biofuels have to demonstrate a 35% (later increasing to 50/60%) saving in life-cycle GHG emissions. This could be problematic in the World Trade Organization (WTO), as a non-compliant biofuel with a 34% emissions saving would probably be judged to be ‘like’ a compliant biofuel. A more economically rational way to reduce GHG emissions, and one that might attract greater public support, would be for the RED to reward emission reductions along the lines of the FQD. Moreover, this modification would probably make the provisions more acceptable in the WTO, as there would be a clearer link between policy measures and the objective of reductions in GHG emissions; and the combination of the revised RED and the FQD would lessen the commercial incentive to import biofuels with modest GHG emission savings, and thus reduce the risk of trade tension.

Shoot yield drives phosphorus use efficiency in Brassica oleracea and correlates with root architecture traits

The environmental and financial costs of using inorganic phosphate fertilizers to maintain crop yield and quality are high. Breeding crops that acquire and use phosphorus (P) more efficiently could reduce these costs. The variation in shoot P concentration (shoot-P) and various measures of P use efficiency (PUE) were quantified among 355 Brassica oleracea L. accessions, 74 current commercial cultivars, and 90 doubled haploid (DH) mapping lines from a reference genetic mapping population. Accessions were grown at two or more external P concentrations in glasshouse experiments; commercial and DH accessions were also grown in replicated field experiments. Within the substantial species-wide diversity observed for shoot-P and various measures of PUE in B. oleracea, current commercial cultivars have greater PUE than would be expected by chance. This may be a consequence of breeding for increased yield, which is a significant component of most measures of PUE, or early establishment. Root development and architecture correlate with PUE; in particular, lateral root number, length, and growth rate. Significant quantitative trait loci associated with shoot-P and PUE occur on chromosomes C3 and C7. These data provide information to initiate breeding programmes to improve PUE in B. oleracea.