A Model Ocean To Study Nitrogen Use

A review of: Bragg, J.G., Dutkiewicz, S., Jahn, O., Follows, M.J., Chisholm, S.W., (2010), Modeling selective pressures on phytoplankton in the global ocean, PLoS ONE, 5, 3, 9569

As our understanding and knowledge of marine microbes improves and increases, new ideas and hypotheses arise to further learn about this vast topic. However, one area that has proven difficult is in the development of a model approach capable of representing the numerous and complex processes that are present in the marine microbial systems. These processes include physical, biogeochemical and biological forces, which can influence such factors as microbial growth, population dynamics and ecological processes such as predation and species interactions. These processes are addressed in this study by the incorporation of a global numerical simulation to study marine pictophytoplankton for nitrogen use abilities. 

This model is created using the marine pictophytoplankton Prochlorococcus and Synechococcus which are dominant planktons in tropical and subtropical ecosystems respectively. Within each of these genera, are many ‘ecotypes’ consisting of populations with different habitats and nitrogen using abilities. Examples of these include some of the Synechococcus ecotypes abilities to use nitrate, nitrite and ammonium as nitrogen sources. Many Prochlorococcus ecotypes do not possess the ability to use nitrate. 

The results of this model show that tropical regions are often dominated by picophytoplankton and higher latitudes are dominated by large phytoplanktonic groups. All of the phytoplankton used in the model could originally use nitrate, nitrite and ammonium, however as the model ran, mutations occurred and produced three different types of phytoplankton, one unable to use nitrate (M1), the second unable to use nitrate and nitrite (M2) and the third was a ‘null mutant’ resembling the parent in all respects (M3).

The loss of nitrogen use abilities was witnessed to occur at different biogeographic regions depending on the organism. One of the main observations seen was the lack in abundance of the M2 mutants at higher latitudes, indicating that they were disadvantaged in these regions, this indicates that being able to use nitrogen in these regions is an extremely important ability. This results in a higher selective pressure against the M2 mutants due to the higher latitudes containing larger quantities of inorganic nitrogen.

This is a very important test regarding the ocean processes that involve marine microbes, due to the application of studying selective pressures on functional traits. The results found tend to highlight the importance of studying the physical, biogeochemical, ecological and evolutionary processes that affect microbes in the marine environment. As this is a model, there are many ways to make it a more comprehensive test, allowing for a vast amount of  further research in this area, particularly in that of the effects of differing latitudinal gradients on microbial functional traits. It also allows the marine environment to be studied in one go, which is vastly beneficial to the understanding how the differing populations of microbes survive and flourish.

Symbiotic Degradation Of Crude Oil

A review of: Hii, Y.S., Law, A.T., Shazili, N.A.M., Abdul-Rashid, M.K., Lee, C.W., (2009), Biodegradation of Tapis blended crude oil in marine sediment by a consortium of symbiotic bacteria, International Biodeterioration & Biodegradation, 63, 142-150

As the human population increases, so does its demand for fossil fuels, this raised demand has lead to larger, and more frequent transportation modes for distribution, one of the most widely used is in large ocean tankers. The increased traffic of these leads to an increased risk of oil spills in the marine environment. The spillages of oil in the world oceans is a huge threat to the marine habitat and the organisms in it. The most affected part of the habitat due to oil spills is in the sediment, and considering the importance of the habitat provided by marine sediments, this is very problematic. Oil also has a longer residential time in sediments when compared to water, and can pose long-term hazards to marine life.

The aim of this study is to provide information on the consortium of bacteria to combat oil pollution in the environment. This will be done by determining the identity of bacteria that are responsible for degrading blended crude oil. Also determining the effectiveness of the bacteria in the degradation of higher molecular weight hydrocarbons. The test was carried out by preparing flasks containing 100g sterilized sediment material along with 1g of crude oil, and adding to them the bacterial population. Bacteria were identified using direct 16S rDNA sequencing. Data analysis consisted of non-parametric analysis when data was not normally distributed and parametric analysis when data was normally distributed. A biodegradation rate was formulated and these were tested using an ANOVA test and Tukey’s test.

The results show that, under optimal conditions, the bacteria responsible for the degradation of the crude oil, P. pseudoalcaligenes, degraded 583.3mgkg^-1 of the blended crude oil from 1000mgkg^-1 oil contaminated sediment over a period of 10 days.

The identified bacteria consisting of P. pseudoalcaligenes, was found to drastically increase the degradation rate of the blended crude oil in the sediment. A symbiotic relationship was observed between the P. pseudoalcaligenes and E. citreus when the two populations are present, the degradation rate is further increased and allowed for the degradation of polycyclic aromatic hydrocarbons. These symbiotic relationships can be used to formulate bacterial consortias for degradation of crude oil compounds, these can be mixed with other isolated bacteria from different locations, however the symbiotic relationships are highly species dependent and will not be universal.

Marine Microbes as Pharmaceutical Agents

A Review of: Waters, A.L., Hill, R.T., Place, A.R., Hamann, M.T., (2010), The expanding role of marine microbes in pharmaceutical development, Current Opinion in Biotechnology, 21, 780-786

Marine microbes are a phenomenally vast group of organisms which cover many of the niches in the marine environment. These organisms are a major focus for drug discoveries and bioactive metabolites. One major area of existing study is that of invertebrate symbionts as creators of molecules that are beneficial to their hosts. This ability is desired for the creation and synthesis of molecules that are beneficial enough to enter clinical trials. Another promising area of study is that of toxins found in harmful algal blooms (HABs), which are increasing in frequency and as a result are becoming a serious hazard to the marine environment and humans alike. 

HABs are a major toxicological issue and can affect many organisms due to the build up of toxins in tissues. With 50,000-500,000 incidents per year, the biotoxins found during these blooms have been affecting humans with a 1.5% mortality rate, they also pose many economic problems due to tourism and fishing industries, an estimated $82 million per year is lost from the US economy due to the effects of HABs. However, upon studying these biotoxins, it has been discovered that they possess a unique stability in the environment including metabolic stability. This can be advantageous to humans as they could possibly be modified into functional drug compounds. An example of this is the karlotoxins, which are a serious hazard as a HAB but have allowed for the design of cholesterol targeting drugs.

Karlotoxins are produced from a toxic suite of metabolites from the dinoflagellate K. veneficum. Due to their unique properties, karlotoxins are able to be synthesised into a non toxic cholesterol pharmacore that has the potential to transport cholesterol from the arteries and into the liver or kidneys for excretion. If effective, this would result in a decrease in serum cholesterol levels and a large increase in HDL cholesterol levels. Due to current research linking cholesterol to numerous human health issues such as cancer, HIV-1, Alzheimer’s and Parkinson’s diseases, this is a very important area of study.

Marine microbes provide a great insight into what is possible for the future of human health advancements, with a multitude of uses, they may soon be essential to pharmacology. This is primarily due to their ability to produce unique compounds which can have multiple roles, these compounds cover new chemical space and allow for substantial growth in the pharmaceutical pipeline. Marine microbes also allow for improved methodologies in fermentation, biosynthesis and synthesis, allowing for new drugs to be created and supplied. Biotechnological pharmacology is certain to benefit from the use of marine microbes as both ways to make new drugs, and sources of unique chemicals that can be used in them.

The Many Isolates of VHSV In Rainbow Trout

A review of: Campbell, S., Collet, B., Einer-Jensen, K., Secombes, C.J., Snow, M., (2009), Identifying potential virulence determinants in viral haemorrhagic septicaemia virus (VHSV) for rainbow trout, Diseases of Aquatic Organisms, 86, 205-212

Viral haemorrhagic septicaemia virus (VHSV) is virus responsible for the disease viral haemorrhagic septicaemia, a disease responsible for a large loss of the rainbow trout Oncorhynchus mykiss. So far, four main genotypes have been identified which allows for genomic regions to be made for analysis. Until recently the rainbow trout that were infected by VHSV were isolated to the geographic location of the Genotype I, but due to the sourcing of food fed to farmed fish, there are many contaminations that have occurred leading to infections of other genomic regions.

The aim of the test is to determine the mortality of the four different genotypes and to analyse the sequencing data. This was done by isolating 24 fish per study group then infected with the different strains of virus intraperitoneally. A control was set up containing an inoculum. RNA was then extracted using a viral RNA kit, cDNA was then synthesised from the viral RNA using a polymerase kit. Polymerase chain reactions were then conducted on each sample to give a consensus nucleotide sequence per isolate.

Results of the mortality test show that the SE-SVA-1033 (GIb) isolates had a mortality of 75% with the majority of fish dying at between 6 and 10 days after exposure. The other three strains resulted in mortalities of 34.6% and under. These deaths also occurred at the same point as those exposed to the SE-SVA-1033 strain but the mortality was reduced. There was no mortality in the control group allowing for the test to be validated. Results of a Chi-squared analysis revealed a statistically significant difference in the mortality rates between treatments. A complete coding region for VHSV strains SE-SVA-1033 and DK-4p37 were also determined.

The results of the infection trial confirm other studies carried out on the mortality of the trout when immersion tested with the SE-SVA-1033 isolate. Observations were made when looking into the lower mortality rate isolates when inoculated, whereby the fish had significant lower mortality rates which indicates that these isolates are less  virulent. 

Further studies can be based on the findings of this test, mainly further looking into the amino acid substitutions that were identified across the genome. This change clearly has an effect upon the virulence and mortality of the virus to the rainbow trout. The exact significance of these mutations can be studied in order to view possible causes to the virulence of the VHVS isolates and as more occur, these can be cross referenced with the genomic data found.

Coral Reefs - More Danger Than Originally Thought?

A review of: Mao-Jones, J., Ritchie, K.B., Jones, L.E., Ellner, S.P., (2010), How microbial community composition regulates coral disease development, PLoS Biology, March 2010 Issue, 44-51

Coral reefs are one of the most delicate and fragile ecosystems on the planet, they are also the habitats that hold over a quarter of all marine species. These two facts result in them being of huge importance to the marine system as a whole, with one small problem facing the symbiotic dinoflagellate zooxanthellae leading to catastrophic effects to the entire ecosystem, it is for this reason that corals are constantly monitored for chemical, physical and microbial change.

Microbes pose a huge threat to corals, with infectious diseases being a leading cause for the increased worldwide coral reef decline. The effects of these diseases are worsened when coupled with the dangers of coral bleaching, caused by an increase in ocean temperatures, which can actually make corals more susceptible to microbial disease. The wide use of the Vibro spp. in tests has been extremely beneficial to understanding the risks posed by disease and possible ways to reduce this risk.

This study looks into using a model to test how the Vibro spp. in corals has an effect on spatial gradients, production of a mucus layer and the abundances of other microbial life. The model used consists of two main rules, that microbial populations are measured in units of growth limiting substrate, and that there is no external inoculation. Once these have been made, the model uses the abundance of pathogenic microbes and the abundance of antibiotic producing beneficial microbes plus antibiotics and substrate.

Results show that models can show insights into the problems that corals face, a rising temperature of oceans, causing an increased susceptibility to pathogens. There can be two states that corals face microbial, and these are the domination of pathogens, and the domination of beneficial microbes, the model can help predict changes that can occur in the balance between these two states due to a number of factors, such as an increase in temperature. However, findings of the model indicate that even if the stress of heat is removed, the corals susceptibility to pathogens can remain and this shift in balance is enough to rid the coral of the beneficial microbial community. This model is highly dependent on the assumptions of the beneficial microbes antibiotic abilities, which can cause unexpected shifts in the outcomes of the models. This is mostly due to the model being carried out int he SMC which can host much more microbial life than the natural ocean. Results show that a shift between the two states is much more favorable to go towards the direction of the pathogen as opposed to the beneficial microbes.

As this model allows for numerous factors to be tested, including factors that directly affect coral susceptibility such as poor water quality, an increase in temperature and poor quality of habitat, it is a very good model for investigating the effects of pathogens on the beneficial microbes that allow the corals to survive.

The Role of Marine Viruses and the Understanding of Nutrient Cycling

A review of: Brussaard, C.P.D., Wilhelm, S.W., Thingstad, F., Weinbauer, M.G., Bratbak, G., Heldal, M., Kimmance, S.A., Middelboe, M., Nagasaki, K., Paul, J.H., Schroeder, D.C., Suttle, C.A., Vaque, D., Wommack, K.E., (2008), Global-scale processes with a nanoscale drive: the role of marine viruses, The ISME Journal, 2, 575-578

Viruses represent the largest pool of genetic diversity and are by far the most numerous of all biotic agents on earth, with an estimated number of around 10^30 viruses in the ocean, they can take over cells in organisms varying from bacteria to sharks, to humans. However, even given this data, the extent of viruses in nanoscale processes are rarely linked to global scale biodiversity and biogeochemistry.

The poor connections between viruses and biogeochemistry is mostly due to the processes being ignored in most carbon flux models. Although much of this data is as of yet unknown, there is much room for research into completing comprehensive models including it. However, due to the findings that they accelerate the recycling of growth limiting nutrient elements in the photic zone, should they be measured as a hinderance or a stimulant to the primary production. There is much ongoing debate as to whether viruses short circuit the biological pump by releasing elements back to their dissolved phase, whether they prime the biological pump by accelerating host export from the euphotic zone, or whether they drive particle aggregation and transfer of carbon into the deep sea. They have also been hypothesized to contribute to the resilience of ecosystems.

An area where viruses are moving forward in biogeochemistry is the combination of molecular techniques such as molecular probes and viral gene expression, to allow for biodiversity, biogeochemistry and genomics to come together. With recent developments allowing for the wealth of the information of the viral genetic reservoir to be discovered using genomic tools. Although, while viruses can be key parts of biogeochemical cycles, it is in what way they do this that is an important area for study. Without the deciphering of metaviromes being required for testing, the ‘blueprints’ of the viruses actions are still locked in the viroplankton metagenomic data sets. Resulting in many viruses that are not in the genomic database having unknown functions. This would be greatly helped with a smooth communication between studies of the virus-host systems in marine microbes and viral biogeochemistry.

In future, the role of viruses can be further understood with tests such as the quantitative reverse transcription–PCR, allowing the accurate measurement of messenger RNA and the expression of the genes. Virology, especially in aquatic and marine habitats, will allow for future collection and understanding of data on the links between the virus-host systems and nutrient cycling and energy flow. With the study of a subject that is still highly debated as to whether it is a life-form in itself, is always going to be a difficult challenge, but backed up with current knowledge, there is the potential to expand our knowledge of viruses and their role in the marine environment in future to give us a greater understanding of how the worlds most important systems work.

Rising CO2 Levels - Worse Than You Thought!

A review of: Doney, S.C., Ruckelshaus, M., Emmett Duffy, J., Barry, J.P., Chan, F., English, C.A., Galindo, H.M., Grebmeier, J.M., Hollowed, A.B., Knowlton, N., Polovina, J., Rabalais, N.N., Sydeman, W.J., Talley, L.D., (2012), Climate Change Impacts on Marine Ecosystems, Annual Review of Marine Science, 4, 11 -37

The marine ecosystem is a very large, complex and delicately balanced world process, however, it is primarily driven, and largely made up of primary producing microorganisms. These are essential in all marine food webs and allow for organisms throughout these webs to survive, including humans, however, with an increase in the human population, there is a greater demand for fossil fuels, and as a result, a larger amount of CO2. This rise in atmospheric CO2, is one of the most critical problems facing our planet due to its global and irreversible effects on the environment. It is in the oceans that one of the largest threats is posed, and this is due to the nature of CO2 which can both raise temperatures of the oceans and lower its pH. This rise in temperature can also lead to the the rising of sea levels, with many coastal habitats being lost, including many of the worlds reef systems. The increase in dissolved CO2 also results in hypoxia where oxygen levels haven been lowered, this is vey problematic for higher species, but can be beneficial to some microbes. However, diatoms will suffer from this as literary tests show that the calcification process is severely hindered in the presence of raised CO2 levels. 

One of the largest problems caused by rising CO2 levels is posed to the symbiotic dinoflagellate zooxanthellae found in corals which, when occurring in the tropics, result in a boom in biodiversity density containing one quarter of all marine species. Risks posed by the rising CO2 levels are the acidification preventing normal calcification of the coral, and the rising temperatures causing bleaching due to the highly sensitive nature of the zooxanthellae. So not only is it the coral organisms that are at a very high risk, but the whole ecosystem that is based around and in them is too. Another area of coral reefs that is directly threatened, is that of the coralline algae coverings, forming a biofilm for reproductive purposes and also as a primary producing food source to other organisms.

As the marine microbes are very often the primary producers in a food web, their importance to the whole world process is of dire importance, this becomes evident when looking at the ice dominated polar systems, where a direct effect can be witnessed when zooplankton have a reduced primary production and are seen to have a knock-on effect to many species in the tropic levels above them.

In its current state, the world oceans contain many of the most delicate ecosystems to be found, this is to do with the delicate nature of many of the microbes that dive the main processes found in them. Due to the delicateness of these organisms, they are posed the largest risk by the very small changes that are predicted from the rising levels of atmospheric and dissolved CO2.

Xenobiotic Metabolizing Bacteria of the Indian Ocean - Getting More Common?

A review of: Rodrigues, J., Madhukar, A., Kumar, N., Sangodkar, U.M.X., (2010), Isolation and characterization of a marine bacterium belonging to the genus Alkalignes capable of the complete mineralization of the dibenzothiopene, Indian Journal of Geo Marine Sciences, 40, 3, 391-397

With a rising demand for fossil fuels from the ever expanding human population, there is always a need for the transportation of products between countries, this is most commonly done via tanker on the worlds oceans. But in recent years this method of transport has resulted in the contamination and near destruction of many of the worlds marine ecosystems. In many places around the world this is a huge threat, but considering that there are many areas with a vast concentration of marine life bordered and regulated by developing countries, this threat can be further increased. 

This study directly looks at the western coast of India region of Goa, where, in 1995, the M.V. Sea transporter ran aground during a cyclone, resulting in the spillage of furnace oil. A marine microbe of genus Alkaligenes was found to be live here and was observed to completely mineralize the xenobiotic compound dibenzothiopene (DBT), an aromatic hydrocarbon found in the tar balls of the contaminated site. The purpose of this test is to look directly at the relationship between the Alkaligenes and the DBT.

During the study, sequential enrichment techniques were favoured, whereby cultures of the bacteria were grown in artificial sea water and to this, 1g of DBT was added and mixed thoroughly to simulate conditions found at the site of contamination. Cultures were then acidified at different times and extracted using an ethyl acetate extract and thoroughly dried. Preparative silica gel G was used to apply the extracts to TLC plates and then exposed to UV light. Residual chemicals were then compared using  industry standard chemicals. The different strains were then separated and tested further against the DBT.

Results show that there are 5 strains of the bacteria that utilizes the DBT, and out of these, the JR110 strain. This was then sent to the IMTECH facility in Chandigarh, India for detailed testing with results as follows, the JR110 strain was seen to degrade 8 different aromatic compounds also with degradation of DBT intermediates to form a red pigmented colony. Tests carried out exposing only the JR110 strain to the DBT were largely successful, showing a clean sigmoid curve of reduced contamination as the growth of the bacteria increases.

Conclusions are drawn that due to the highly contaminated nature of the west coast of India, there is likely to be continuous evolution of degradation processes of xenobiotic compounds, which is believed to be due to the intermediates of benzoate utilizing the pathways in marine bacteria, thus exerting a positive pressure on the mineralization of xenobiotic compounds such as DBT.

Monitoring sewage pollution with sea fans

A review of: Baker, D. M., Jorda’n-Dahlgren, E., Maldonado, M. A. and Harvell, C. D. 2010. Sea fan corals provide a stable isotope baseline for assessing sewage pollution in the Mexican Caribbean. Limnology and oceanography, 55(5), 2139-2149.

As populations continue to rise, especially on the coastlines, sewage pollution is becoming a greater threat to coastal marine systems. The input of contaminants, especially nutrients such as nitrogen and phosphate can greatly disturb the environment. Increases in primary productivity can smother critical species such as sea grasses and reef building corals, pathogens can cause disease especially in corals and there is a generally common result of lowered biodiversity. While monitoring of this kind of activity is important everywhere, it is especially important that it is studied and controlled in developing regions which are often dependent on their ecosystem’s health and wellbeing.

This study hypothesised that sewage-derived nitrogen inputs are detectable and more severe in developed areas along the Mesoamerican barrier reef of Mexico. To test their hypothesis in this area they compared the stable nitrogen isotope (δ15N) values from the common Caribbean sea fan, Gorgonia ventalina, collected from a developed and undeveloped are of the coastline. Akumal coast was selected as the developed site as there are a great number of residents and a huge influx of tourists. The shoreline of Mahahual was selected to be the undeveloped site since there were few residents and tourists and a sewage treatment infrastructure.

The isotopic ratio of 15N:14N is regarded as an effective and direct indicator of human nitrogen pollution. Enriched isotope values arise from the accumulation and degradation of human and animal wastes and are easily distinguishable from other sources. Perpendicular to the shore samples of sea fans were taken 1km from the shore. 2cm-squared fragments were cut that are
likely to represent the previous year of growth. Stable isotope analysis was performed on the samples. The prevalence of Enterococcus was sampled at sites adjacent to where the sea fans were sampled. Enterococcus assays were used to determine if the nitrogen isotope analysis values were correlated with sewage pollution. Positive results for this test would rule out the possibility of enrichment due to denitrification. Statistical analysis was then performed.

Results found that samples from the developed site sea fans were enriched in δ15N (as high as 7.7‰ near shore) and were ≈3.5‰ greater than sea fan samples from the undeveloped site. The δ15N values were also positively correlated with faecal Enterococcus counts from the seawater. This confirms that the enrichments are associated with sewage and not denitrification. This study suggests that data collected from the undeveloped site which is relatively pristine could now be used as an isotopic baseline for monitoring the Mesoamerican barrier reef at sites where increased development is planned or underway. Another interesting find of this study was that the highest Enterococci counts were found to be from a lagoon popular with bathing tourists. The counts were approximately 59CFU per 100mL. The presence of faecal Enterococci is well above US Environmental Protection Agency standards for recreational waters which is 35CPU per 100mL.

This method of stable isotope analysis in sea fans could be a promising tool for monitoring changes in the contribution of human nitrogen sources to nearby ecosystems, especially in developing regions where water quality monitoring programs are not established.

Not just for the Bathroom...

Marine sponges are well known for harbouring a wide variety of microbes which have been found to have a mixture of roles, including protection, pathogens and competitors.  Sponges are also the most prolific marine producers of novel compounds, many of which are of pharmaceutical and biotechnological importance. The structural similarity between the compounds found in sponges and compounds found in sponge microbiota suggest that they could in fact be of microbial origin. Sponge-associated microorganisms have recently received renewed attention, with much research focusing on the production of bioactive compounds. This research has found that bacteria belonging to the actinobacteria genera are the largest producers of these secondary metabolites.

The aim of this investigation is to analyse the compounds produced by bacteria from two species of sponge (Suberites carnosus – non-calcarious, and Leucosolenia sp. - calcarious) in an effort to produce novel antibacterials to combat drug resistant pathogens, which are becoming a big problem in the medical industry.

Samples of the sponge species were collected at a 15m depth from Lough Hayne, Co. Cork in November 2008. Bacteria from these samples were cultivated on agar over a period of two months, during which time they were incubated at 18^oC and checked regularly for distinguishable colonies.  After the incubation period, the cultivated bacteria underwent deferred antagonism and well diffusion assays, PCR and phylogenetic analysis of the 16S rRNA gene.

For both sponge species, analysis of 16S rRNA sequences revealed that the dominant phylum of bacteria found was Protebacteria, and the most abundant in both cases was γ – Proteobacteria. The genetic identity of over 98% of the isolates found in both sponge species are already known to science. However, some isolates, especially from Leucosolenia sp. are not. A result like this is not unexpected, as calcarious sponges have been subject to little research before now. A wide range of the bacteria found in both sponge species displayed antimicrobial activity during analysis, though more of the isolates from the sponge species S. carnosus showed this than those from Leucosolenia sp. This can be explained by different genera and species isolated in both, as the dominant genera of bacteria found was different in each species of sponge (Pseudoalteromonas and Vibrio in Leucosolenia sp., and Pseudovibrio and Spongiobacter in S. carnosus).

The authors summarised that in both sponge species, a high level of anti-microbial activity was found. Leucosolenia sp was found to produce more effective antifungal compounds, whereas S. carnosus appeared to contain more microbes with antibacterial properties. The researchers did struggle to isolate the compounds responsible for this activity in many cases however, meaning that the paper lends itself easily to further research. Overall, the findings from this paper may prove to be useful in future, particularly to the medical industry.

A review of Flemer B., Kenedy J., Margassery L.M., Morrissey J.P., O’Gara F. and Dobson A.D.W. (2011) Diversity and Antimicrobial Activities of Microbes from Two Irish Marine Sponges, Suberites carnosus and Leucosolenia sp., Journal of Applied Microbiology, 112, 289-301

Nitrification in the OMZ of the Arabian Sea (OMG!)

A review of: Newell SE, Babbin AR, Jayakumar A, Ward BB (2011) Ammonia oxidation rates and nitrification in the Arabian Sea. Global Biogeochemical Cycles, 25: 1-10.

A major rate limiting nutrient in the marine environment is nitrogen and oxidation of ammonia derived from organic matter is postulated to support 12-32% of primary production globally. The involvement of ammonia oxidising archaea (AOA) from the phylum Crenarcheota in nitrogen recyling was a relatively recent discovery. It was previously thought that ammonia oxidation was restricted to groups of β and γ proteobacteria (AOB), however more recent studies reveal AOA numbers that are considerably higher than AOB. The rates of archaea and bacteria ammonia oxidation in the oceans have not been quantified separately and archaea metabolism in the mesopelagic zone is yet to be fully elucidated. The Arabian Sea oxygen minimum zone (OMZ) is the largest in the world and the nitrogen cycle within this body of water has been the focus of many recent studies. The aim of the investigation by Newell et al (2011) was to explore the factors that influence the rates of ammonia oxidation and the abundance of AOA and AOB on the surface of the Arabian Sea, in low oxygen regions and in the mesopelagic zone below the OMZ.

Specific primers to identify the gene that encodes the ammonia monooxygenase enzyme (amoA) were used to estimate abundance of AOA and AOB using qPCR. Bacteria and archaea amoA genes were detected at every sampling depth in three locations, revealing AOA to be 35 to 216 times more abundant than AOB. Ammonia oxidation rates ranged from undetectable to 21.1 ± 0.1 nmol l^-1 d^-1; with the highest measurement near the surface layer (40 to 80 m in depth). The rates of ammonia oxidation within the oxycline (95-136 m in depth) above the OMZ were between 1.5 ± 0.2 and 4.6 ± 0.4 nmol l^-1 d^-1, while a low average of 0.07 ± 0.05 nmol l^-1 d^-1 was detected  in the mesopelagic zone (900 to 1000 m in depth)directly below the OMZ.

AOA appear to be the dominant ammonia oxidisers in the Arabian Sea, significantly contributing to nitrite production within the primary nitrite maximum (PNM) zone above 100 m and may be responsible for 10-100% of nitrite supply required to maintain the PNM. The role of ammonia oxidising Crenarchaeota in the deep ocean remains unclear however amoA gene abundance of 10⁵ copies ml^-1 at around 1000 m indicates a large population that substantially impacts nitrogen and carbon cycling. AOA nitrification in the photic zone provides recycled nitrate for primary production and may be a significant source of nitrous oxide (a greenhouse gas) which warrants further investigation. The role of AOA in OMZs and the metabolic pathways that sustain growth in hypoxic and anoxic conditions also requires elucidation, along with microbial diversity (via 16S gene analysis) and community dynamics. Furthermore, the abundance of amoA genes in the mesopelagic zone suggests approximately 50% of microbial biomass could be autotrophic and nitrification may be the main process that utilises most of the ammonium produced from organic carbon fluxes.         

Bioactive potential of Seagrass bacteria against human bacterial pathogens

The discovery of drugs from natural sources is an increasing trend. Researchers are searching for more novel chemical entities, in the hope they will provide new leads for disease treatment. The marine environment is particularly interesting for researchers because of the harsh physical and chemical conditions experienced by many organisms; yet despite this and the biodiversity of the oceans far outweighing terrestrial biodiversity research in this area remains in its infancy.
In particular, marine plants are known to produce a large number of structurally diverse secondary metabolites. Seagrasses are the only angiosperms to successfully grow in sub-tidal and tidal conditions. Several species of Seagrass have obligate microbial populations within their roots, leaves and rhizomes. Some medicines and chemicals are already prepared from Seagrasses and their associates.
An even greater concern than bacteria which are resistant to a single antibiotic is bacteria which are resistant to multiple antibiotics. As antibiotic resistance has developed, researchers have developed alternative antibiotics and combination therapies. However, the constant overuse of antibiotics in humans and their livestock has led to many bacteria being resistant to many antibiotics. The problem of resistance demands renewed efforts to seek antibacterial agents effective against pathogenic bacteria.
Two species of Seagrass from the SE coast of India were sampled and returned to the lab under sterile conditions, for the isolation of epiphytic and endophytic heterotrophic bacteria. 32 strains of endo/epiphytic bacteria were tested for their antagonistic activities against 5 antibiotic resistant human pathogens, of these, 10 were found to be antagonistic against one or more human pathogens. The authors go on to discuss the minimum inhibitory concentrations (MIC) as well as minimum bacterial concentrations (MBC) for the 32 tested strains.
The outcome of this research is that the endophytic bacteria isolated from the Seagrasses showed maximum sensitivity against several of the human pathogens compared with the epiphytic bacteria. And also that the bioactive compounds from the endophytic bacteria show maximum sensitivity with MIC than the bioactive compounds from the epiphytic bacteria.
From the outcomes of this research steps have been taken to find out the reason for the maxium activity of endophytic bacteria from Seagrasses.

There were two main reasons I choose to review this paper 1) The use of natural sources in the fight against humans pathogens is something that I find interesting and 2) In another module I have studies Seagrasses and like to see an over-lap/relation between modules. That being said, I found this paper very hard to read. It appears to me that the authors first language is defiantly not English, the paper did not read smoothly and the sentences seemed very disjointed, this I feel should have been addressed further. There were also seemingly simple mistakes made, for example species names were not italicised – I think these should have been picked up on. Simple changes would have made for a better read.
Despite this, the results of the paper are encouraging and the above mentioned points shouldn’t distract from the outcome. I hope that more antibiotics which can be used against both human and animal pathogens are found, as the problem of antibiotic resistance does appear to be rising.

A review of:
Ravikumar, S., Thajuddin, N., Suganthi, P., Inbaneson, S. J. and Vinodkumar, T. (2010) Bioactive potential of seagraa bacteria against human bacterial pathogens. Journal of Environmental Biology. 31 387-389.

Biofilms lead the way

Biofilms are thought to stimulate the attachment of invertebrates and algae to marine surfaces. This is known as biofouling, which refers to the accumulation of organisms and biogenic structures on marine surfaces. In particular sessile organisms compose the fouling assemblage. Many organisms such as barnacles and tubeworms produce shells and other firm structures during growth, which allow for the attachment of other organisms. This results in a multilayered fouling community. A central issue to the build up of biofouling on ships is an increases fractional drag, biofouling covers oceanographic equipment, coats floating structures and promotes structural deterioration. Biofouling costs industry billions of dollars per year due to prevention costs, maintenance and additional fuel consumption. Biofouling begins with the formation of biofilms followed by the aggregation of other diatoms and other micro-organisms bound together by extracellular polymeric substances. Biofilms can form within hours of immersion and rapidly increase in density and structural complexity. Furthering this, algae and invertebrates and aggregate. Biofouling is thought to be facilitated by the detection of appropriate sub strata and adhesion of larvae. It is thought that chemical cues play a pivotal role in invertebrate settlement. Microbial biofilms produce chemical signals that attract settlement. Antifouling research has provided information about the structure and function of biofilms, in particular with reference to understanding the adhesion and settling of invertebrate influence by microbial films. Zardus et al.'s research investigates the influence of microbial films on the adhesion of newly settled invertebrate larvae, they compared the removal rate of settlers from glass surfaces with and without natural biofilm coatings after exposure to controlled forces of shear. Their research was carried out on four marine fouling organisms: polychaete worm Hydroides elegans, barnacle Balanus Amphitrite, bryozoan Bugula neritina and a tunicate Phallusia nigra.

Larvae of the four invertebrate species were obtained from field collected adults and cultures were maintained in the laboratory following standard protocols. Trials were carried out in a turbulent channel flow apparatus and replicates of treatments were tested on the different invertebrates.

Larvae settlement was much greater on biofilm glass than on clear glass surfaces for Hydroides elegans, same was the case for Balanus amphitrite. Biofilms also had a positive effect on the adhesion strength for some of the settlement stages. Therefore bioflims increase invertebrates shear strength.

Adhesion of larvae is facilitated by microbial biofilms providing a connection between biofouling and biofilms. The mechanisms that cause this relationship require further research. Invertebrates use viscoelastic gels that have an adhesive nature in the presence of biofilms and form complex interactions, the adhesive strength is dependent on the taxa involved. It is suggested that biofilms may also stimulate increases and decreases in the adhesive produced by invertebrates, which has consequences for its structural strength. The trend observed was that with age invertebrates showed stronger adhesive forces. This discovery that biofilms facilitate invertebrate adhesion requires further research. Investigations are called for into the physical mechanisms of the biofilm invertebrate connection, paying particular attention on how these adhesives are modulated.

It is understandable that biofouling is undesirable for ships, however if structures such as marine renewable energy devices attract biofouling, this may have trophic food chain benefits supporting local biodiversity. This may be a desirable outcome provided biofilms don't cause any structural corrosion.

References:

Zardus, J. D., Nedved, B. T., Huang, Y., Tran, C. & Hadfield, M. G. (2008). "Microbial biofilms facilitate adhesion in biofouling invertebrates." The Biological bulletin 214: 91-98

Sorry Gastroenteritis. Theres just no relationship between us. From Entrococci

The purpose of this study was to evaluate the risk of exposing bathers to human pathogens in sub tropical recreational marine waters with non-point source of sewage and other pollutants. The authors also examined possible relationships between microbial densities and random symptoms in human subjects by questioning bathers at random and following up with microbial monitoring.

A group of regular adult bathers were recruited and divided into two separate groups : bathers and non bathers. Bathers were required to spend 15 minutes in knee deep water across the beach and submerge their heads every 5 minutes. Water samples were also collected by the bathers which were assayed for enterococci by membrane filtration. The non bathers group were not allowed to enter the water and were restricted to sitting on a plastic seat in a covered roped off area distant from water and sand exposure for 15 minutes at a time. This was followed up 7 days later with a phone questionnaire to determine if any of the bathers and non bathers had any illnesses.

From the water sampling it was determined that all bathers were exposed to an average of 71 enterococci/100ml of water however there was a very large range between samples. Symptoms reported after a week included gastrointestinal, skin, eye and ear infections which would normally be associated with exposure to contaminated bathing waters. Bathers reported more gastrointestinal, respiratory and skin infections compared to the non-bathers group and also reported a significantly shorter onset time of illness.

This paper confirms that people bathing are more likely to acquire infections from subtropical recreational marine waters compared to those that do not bathe even when there is no source point.

It was also found that the skin illnesses reported were dose dependant, as expected, however there was no relationship between the degree of exposure to enterococci and severity of the respiratory and gastrointestinal infections with the causative agents unknown. This could have further implications as, currently, many U.S. authorities monitor water systems using gastrointestinal illnesses as an indicator to water quality. Therefore, if there is no direct correlation between dose and outcome of gastrointestinal illness, the advice given by these authorities may not be completely accurate.

Although this was the first paper of its kind, all the participants were previously healthy adults and does not represent children or anyone with a compromised immune system.

A review of: Fleisher, J.M., et.al.; 2010; The BEACHES Study: health effects and exposures from non-point source microbial contaminants in subtropical recreastional marine waters; Int. J. Epidemiol.; 39(5): pp1291-98.

Rhamnolipid, another application

I have spoken about the uses of rhamnolipids, a biosurfactant, before and this paper goes on to talk about their potential to disrupt biofilms from surfaces exposed to the marine environment, a simple but generally unexplored area. Specifically this paper focuses on the biofilm crated by Bacillus pumilus and the effect of rhamnolipid on its ability to adhere to steel. Many Bacillus species are resistant to harsh environmental conditions such as low nutrient availability and UV radiation and this hardiness also means they are able to resist anti- fouling biocides. Adhesion to steel and the production of sticky exopolysaccharides and organic acids by Bacillus biofilm leads to increased corrosion and thousands of dollars’ worth of damage. Primary colonizing bacteria are the first to adhere to such surfaces and so the killing and ideally the removal of such is an important problem in need of a solution, biosurfactants could act as that solution.

B. pumilus were allowed to adhere for 4 hours at 30°C. Different concentrations (0.05 – 100mM) of rhamnolipid were then added to the polystyrene microtitre plates and left for a further one hour. After staining the results were taken as a percentage of cell adhesion compared to control plates which were not treated with rhamnolipid.

Exopolymeric substances (EPS), which have been investigated in other blogs, are the basis for adhesion of cellular substances to surfaces underwater. They allow for the attachment of other species leading to a community of microbes ultimately creating a damaging biofilm. Rhamnolipids have been shown to have antimicrobial as well as surfactant properties against other bacteria such as B. subtilis and Staphylococcus epidermis at low minimum inhibitory concentrations (<1.6 mM MIC) however the growth of B. pumilis in this case was not inhibited until >1.6mM and therefore higher concentrations of rhamnolipid are needed compared to other Gram negative bacteria. In contrast to this, at low concentrations, rhamnolipid significantly inhibited the adhesion of 46 – 99% of B. pumilus, after one hour there was at least 80% inhibition of adhesion to the polystyrene surfaces which may be enough to prevent the formation of biofilm. Other tests with different bacteria also show the effectiveness of rhamnolipid as an anti – adhesive when it comes to biofilm.

EPS is thought to neutralize antimicrobial agents and therefore assist the microbial community form dangerous biofilm. Therefore the disruption of such EPS could stop such formation. At higher concentrations than the MIC value there was a significant dose – dependent increase in biofilm disruption after treatment with rhamnolipid. After one hour there was significant EPS disruption and 24hours treatment led to destruction of microcolonies. One problem with this report is the suggestion that rhamnolipid usage would be more successful with the help of biocides. In the marine environment the use of biocides in unattractive due to other environmental hazards this brings about. However, the treatment of rhamnolipids does have potential and should be investigated further.

Review of Dusane et al (2010) Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms

Fugu no longer toxic!

A review of: Noguchi, T., Arakawa, O. and Takatani, T. 2006. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 1(1), 153-157.

Marine pufferfish of the family Tetraodontidae can be known to possess a neurotoxin (TTX/Tetrodotoxin). This toxin is poisonous to humans, causing paralysis and even death. In Japan ‘Fugu’ is a popular dish which is made from these pufferfish. Many believe the liver to be the tastiest part, however this is also the most toxic. Safe preparation of this dish requires a great amount of training and the serving within restaurants is strictly controlled. The serving of the liver has even been banned due to a frequent occurrence of food poisonings. No detailed studies have been made on the mechanisms of uptake, accumulation, metabolism and excretion of TTX in pufferfish. While they do have a TTX-secreting gland or cells within their skin which is thought to be a defence mechanism, only a small amount if any is ever produced and this does not intoxicate the liver. Previous studies have suggested that the pufferfish do not synthesise the TTX by themselves. Instead they are thought to accumulate it through the food chain. So this study hypothesised that non-toxic pufferfish should be able to be produced if cultured with a TTX-free diet.

Over 5000 species of the pufferfish, Takifugu rubripes were cultured by netcages at sea, and aquaria on land for up to three years. As they are known to accumulate the toxin mainly in the liver and ovary following ingestion, the liver was focussed on for testing, but other parts were also used. Toxicity was assessed using the Japanese official mouse assay method for TTX and also LC/MS analysis. Each tissue was extracted with 0.1% acetic acid and then examined for toxicity through intraperitoneal injection into male mice.

The results indicated that all parts of all pufferfish tested were considered to be ‘non-toxic’ in both the mouse assay where levels were less than 2MU/g and the LC/MS analysis where levels were less than 0.1MU/g. The expression MU is used where 1 MU is defined as the amount of toxin required to kill a mouse in 30 minutes after injection. Anything less than 10MU/g is considered to be non-toxic in food hygiene. This provides solid evidence that the pufferfish become intoxicated through the food chain and non-toxic fish can be successfully produced by netcage or land culture irrespective of culture area and season. If cultured in this way, the fish and in particular the liver can be considered safe to consume.

Lava Eaters

The aim of this study was to assess the abundance, species richness and phylogenetic diversity of endolithic and epilithic microbial communities inhabiting young, unsedimented ocean crust at the sea floor. Hence, basaltic lavas of various ages and alteration states were sampled by the authors from the East Pacific Rise (EPR) and around Hawaii and then analyzed using quantitative PCR, FISH and microscopy. The PCR measurements of the glassy rinds of lava flows showed that the total bacterial and archaeal cell densities were ranging from 3x10⁶ to 1x10⁹ cells/g and that bacteria were dominating (88–96%) all the rock samples examined. This results were then confirmed by the FISH analysis, which revealed dense populations of Bacteria (significantly more abundant than Archaea) exhibiting cell abundances of 3-4 orders of magnitude greater than in the overlying deep sea waters (8x10³ - 9x10⁴ cells/ml) where half of the cells were instead, Archaea.

Subsequently, in order to evaluate in more detail the community composition, the authors used fulllength 16S ribosomal RNA gene clone libraries constructed from basaltic lavas and surrounding sea water samples. These phylogenetic analyses revealed that both the basalt-hosted biospheres (EPR and Hawaii), were harbouring high-richness bacterial communities and that community membership was shared between these sites. A statistical approach was then used to evaluate the species richness (number of operational taxonomic units) as compared to other oceanic environments analyzed in other studies (e.g. an hydrothermal white smoker, the upper water column of the Sargasso Sea, hydrothermal fluids from the Mid-Atlantic Ridge and deep-subsurface sediments from the Nankai Trough). These comparative analysis, revealed that abundance, phylogenetic diversity and richness of Bacteria in these other deep-sea environments were clearly lower and much different than EPR and Hawaii deep-sea basalts. The 21 taxonomic groups recovered from basalt were dominated by Proteobacteria (68% and 66% of all sequences in EPR and Hawaii respectively), while non-Proteobacteria groups included Plantomycetes (8%/5%), Actinobacteria (7%/8%), Bacteroidetes (4%/1%), Acidobacteria (3%/4%) and Verrucomicrobia (2%/2%). Interestingly, the OTU richness for the two geographically separated basalt communities (EPR and Hawaii) showed considerable overlap in community membership, suggesting that oceanic basalt microbes are widely distributed among this biotope.

These differences in phylogenetic diversity, species richness, and total biomass between the basaltic lavas and overlying sea water raised questions about what energy source fuel this biosphere. Potential energy sources capable of sustaining microbial life in ocean crust include hydrothermal input of manganese and iron (chemolithoautotrophic growth) and dissolved organic carbon in sea water or hydrothermal fluids (heterotrophic growth). However, according to the authors, the most plausible explanation is that oceanic lithosphere exposed at the sea floor undergoes seawater-rock alteration reactions and these reactions are capable of supplying sufficient energy for chemolithoautotrophic microbial growth. Lava surfaces in fact, are composed predominantly of volcanic glass, a highly reactive rock component that contains reduced elemental species such as iron, sulphur and manganese. Oxygen and nitrate in deep sea water oxidize these  constituents and chemolithoautotrophic microorganisms can potentially exploit the free energy changes associated with these redox reactions for their metabolic requirements. Laboratory studies have already demonstrated that iron-oxidizing bacteria isolated from the sea floor are able to use rock and minerals, including glassy basalt, for metabolism and growth. The authors estimated that about 6x10⁷-6x10⁹ cells per g basalt may be supported through these reactions and actually, cell densities in EPR basalts were falling exactly within this range. So in conclusion, alteration reactions in the upper ocean crust may fuel microbial ecosystems at the sea floor, which constitute a trophic base of the basalt biotope, with important implications for deep-sea carbon cycling and chemical exchange between basalt and sea water. This hypothesis supports the understanding of the phylogenetically rich and distinct nature of the basalt biotope. The enrichment of taxa from diverse metabolic groups may result from the establishment of chemical microenvironments within or on rock cavities and surfaces during alteration, mineral precipitation and biofilm formation. This niche creation would allow for a greater variety of redox reactions and metabolic pathways (e.g. heterotrophic, anaerobic, or reductive) including those supporting complex organotrophic and mixotrophic communities. 

Reference:

Santelli, C.M., B.N. Orcutt, E. Banning, W. Bach, C.L. Moyer, M.L. Sogin, H. Staudigel, and K.J. Edwards. (2008). Abundance and diversity of microbial life in ocean crust. Nature 453:653-656.  

New Species of Bacteria Isolated from the RMS Titanic

A review of: Sánchez-Porro, C., Kaur, B., Mann, H., and Ventosa, A. (2010) Halomonas titanicae sp. nov., a halophilic bacterium isolated from the RMS Titanic. International Journal of Systematic and Evolutionary Microbiology 60:2768-2774

A new species of bacterium isolated from the RMS titanic is described in this paper. The bacterium was isolated from the rusticles on the ship, bioconcretious structures that look like icicles but are formed from oxidised iron. Various microorganisms live within these structures, which are the by-product of the microorganisms effectively feeding off the metal of the ship.

In this study, the authors used phylogenetic analysis alongside other methods including obtaining 16s rRNA though PCR to perform a BLAST search in order to try and identify the strain. The results of these methods showed that the strain isolated was most closely related to the genus Halomonas. Its most closely related species included Halomonas neptunia (98.6% sequence similarity), Halomonas variabilis (98.4%), Halomonas boliviensis (98.3%) and Halomonas sulfidaeris (97.5%), amongst others confirming it belonged in the Halomonas genus. However, several important differences, including phenotypic and chemotaxonomic differences features that confirmed the strain was a separate and distinct species not previously identified. The paper goes on to describe the new bacterium in detail.

The genus halomonas is heterogeneous, containing more than 60 species to date and are a member of the salt-loving Halomonadaceae family which are able to grow at salinities of 5 - 10% and who are considered generally non-pathogenic aerobes. The new bacterium is also gram negative, flagellated and motile.The discovery of this new bacterium is of particular interest as it may contribute more to the understanding of the mechanisms of rusticles, how they form etc. It also can have wider implications in understanding how to protect other submerged metal structures such as oil pipeline, oil rigs and the disposal of ships at sea for example.

Additional reference: BBC. (06/12/2010). New species of bacteria found in Titanic 'rusticles'.Available: http://www.bbc.co.uk/news/science-environment-11926932.

Prebiotics help for soybean meal bases, do they work?

Prebiotics like probiotics are used in aquaculture to maintain the health of a species, however prebiotics unlike probiotics are a non-digestible food ingredient which can benefit the host by stimulating the growth and activity of bacteria residing in the colon, promoting a healthier host organism. This study investigates the effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by the Red Drum Sciaenops ocellatus (Linnaeus).

The authors used a recirculation system for the 35 sub adult Red Drum which were fed a control diet containing 40% crude protein, exclusively from menhaden fish meal, 0% lipid, and an estimated available energyof14.6 kJ g-1. Five experimental diets were to be similar to the control diet, but with approximately 50% of the protein supplied by menhaden fish meal and 50% provided by soybean meal. To four of the experimental diets, prebiotics were singularly added at 1% of dry weight in place of cellulose while the basal diet had no prebiotic supplementation. The prebiotics in use for the trial (a mix or individually) were mannanoligosaccharide (MOS), galactooligosaccharide (GOS) and inulin. The faecal matter was used for nutrient analysis.

The results showed that the basal diet mixed with MOS and GOS significantly increased protein (82% for the three tanks) and organic matter (69, 64 & 66% for the three tanks) in comparison to the control diet which showed 69% for protein and 49% for organic matter. However the lipid values were significantly decreased for fish fed with MOS, GOS and inulin (63, 61 & 61%) compared to the control of 77%. Energy values were the same for the fish fed with inulin as the control diet of 54%.

This is the first study to demonstrate that nutrient and energy digestibility of soybean-meal-based diets can be enhanced by prebiotics. The wider significance of this investigation is paramount to replacing a higher percentage of fishmeal in fish diets with soybean and or other protein replacements. The side effects such as gastroenteritis can be treated with probiotics and the protein enhancement can be treated with prebiotics, thus a mixture of both in diets would be the next step in further research.

A review of: Burr, G., Hume, M., Neill, W. H., & Gatlin III, D. M. (2008). Effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by red drum Sciaenops ocellatus (Linnaeus). Aquaculture Research, 39, 1680-1686.

The occurrence of enteric viruses in shellfish

A review of: Suffredini, E., Corrain, C., Arcangeli, G., Fasolato, L., Manfrin, A., Rossetti, E., Biazzi, E., (2008), Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors, Applied Microbiology, 47(5):467-474

Shellfish are considered to be potential vectors of foodborne diseases, due to their accumulation of pathogenic microorganisms through filter-feeding. Norovirus (NoV) is an enteric virus that has been linked to shellfish-associated disease outbreaks and is responsible for 60-80% of human gastroenteritis outbreaks. Similarly, shellfish are linked to the transmission of hepatitis A virus (HAV), and its consumption has been reported in 69% of HAV infected patients. In Italy, there have been numerous cases of these diseases related to seafood consumption registered in several cities.

It has been established that climate has direct and indirect effects on the occurrence of enteric viruses. For example, high precipitation can cause flooding and sewage runoff which are key factors in contamination of coastal water and shellfish harvesting areas.

The aim of this study was to assess the prevalence of HAV and NoV in shellfish harvested in a specific national production area, the deltic area of the river Po (North Italy). They also looked at the effect that environmental factors have on viral contamination in the production area.

A survey was carried out for 1 year on samples of shellfish (mussels and clams), taken every 15days from two areas, A (sea area) and B (lagoon area). Environmental parameters such as temperature, pH and salinity were noted as well as bacteriological analysis (for E.coli and Salmonella) and virological analysis (for NoV and HAV), using various PCR methods.

The results showed no significant differences in environmental paramaters in the two areas, with some predictable fluctuations due to seasonal variations. No Salmonella was detected and E.coli numbers were always below legislation limits. There was also no HAV found in both areas but NoV was detected in 10 of the 120 samples, all from area B. NoV was also present in samples ranging throughout the sampling period, with the majority of positive samples in spring and summer. This led the researchers to conclude that a definite association between NoV clinical cases arising during the winter period and shellfish cannot be established.

They also noted that the increase in viral contamination could be in relation to the flow of the tributary river, which could transport further viruses into the harvesting areas and moreover mixes and lifts the sediment at the bottom of the lagoon where viruses can deposit and survive for long periods.

The study highlights the importance of accurate classification of harvesting areas in assuring the safety of shellfish for direct consumption and is useful in helping to establish suitable prevention techniques, especially after meteorological events.

Novel adenovirus isolated from sea lions

Viruses of the family Adenoviridae have genomes consisting of double stranded DNA. They infect various species of vertebrates, including humans. Adenoviruses were first isolated in 1953 from human adenoids. Two types of canine adenoviruses are well known, type 1 and 2. Type 1 causes infectious canine hepatitis, a potentially serious disease involving vasculitis and hepatitis. Type 1 infection can also cause respiratory and eye infections. Canine adenovirus 2 (CAdV-2) is one of the potential causes of kennel cough. Viral hepatitis associated with adenoviral infection has been previously seen in free-ranging California sea lions Zalophus californianus. However previous isolation of viruses were unsuccessful and identification of this virus was ceased and no specific virus was documented. However because the morphological features seen under the microscope were quite similar to that of canine infectious hepatitis and since the virus has a wide host range, it was thought that perhaps the virus responsible for disease was CAdV-1.

The presence of adenoviral DNA was examined in tissue samples from 2 live stranded California sea lions that were admitted to the rehabilitation facility at Californian Marine Mammal Centre. The two rescued animals died with serious symptoms , first was diagnosed with arteritis , pneumonia and pulmonary haemorrhage. Also eosinophilic intranuclear inclusion bodies (sign of adenoviral infection) were discovered in few organs including lymph nodule and lungs. The other case died from severe viral hepatitis with intranuclear eosinophilic inclusions found within hepatocytes.

Tissues from both animals were first examined under electron microscope and then analysed using PCR with the use of specific primers (sense: 5’-GCG CAC TTA CTC ATC CAT TTC C-3’, antisense: 5’-GCT ATT TCT CCA CGC AGC GG-3’). The virus was isololated and compared with known adenoviruses.

The examination of lymph node from first animal and liver of the second one revealed the presence of adenoviral-like particles (70 to90 nm icosahedral) , within the nucleus of affected endothelial cells .The sera from both animals were negative for antibodies against both CAdV-1 and 2. The PCR to detect CAdV-1 and 2 was also negative. Sequencing confirmed the presence of a fragment of the DNA polymerase gene of a novel adenovirus and comparison of the sequence to known adenoviruses in GenBank showed that this was a novel virus from the Mastadenovirus genus. This virus was similar in 77% to tree shrew adenovirus 1 (TSAdV-1) CAdV-1 (72%) and 2 (74%) however in overall is was treated as independent lineage and species.

This was very interesting study and quite exciting that they managed to find new virus , although the more analysis is needed in order to establish if this virus is a primary pathogen causing death. It is quite surprising that although the symptoms of this disease were quite similar to those reported in many other cases , nobody actually managed to identify this virus before.

A review of Goldstein et al.2011 . Isolation of a novel adenovirus from California sea lions Zalophus californianus. Dis Aquat Org. 94: 243–248.