Tuesday 31 January 2012

Turtle Tumours

When going back over the notes of the marine mammal disease lectures I found a slide on turtle diseases which I don’t think we covered; so I thought it might be interesting to delve a little deeper into the area...


Turtles are susceptible to a tumour-forming disease caused by a fibropapilloma-associated herpesvirus. This results in tumours growing on the turtle’s body and on its internal organs which can interfere with feeding, reproduction, etc. in many species. This paper looked at Green turtles (Chelonia mydas) in particular, which are widely affected by fibropapillomatosis. The disease is thought to be transmitted by contact as it has low genetic variability and mutability, suggesting that the pathogen is quite specific to its host and so presumably could not survive as a free living virus. This has led to the suggestion that other factors could be important in disease development such as environment and diet.

The authors suggest that environment could be particularly influential, as in the Hawaiian study population, the disease was only contracted after the turtles spent time in shallow coastal waters once they reached a certain size. Furthermore, coastal runoff enhanced non-native macroalgae growth, suggesting a link between land use and disease. Therefore the paper aimed to investigate the interplay between land use, environment (i.e. macroalgae), size and disease; as well as trying to identify the correct spatial scale for investigating the disease.


Van Houtan and colleagues focused on the Hawaiian population of C. mydas over 28 years by looking at stranding records and indentifying disease cases. They standardised disease rates by size (as it is a known factor in disease susceptibility) in order to gain a detailed picture of the disease through space and time in each size class. They also characterised land use at each stranding site and calculated the nitrogen footprint of each area.


The study found strong links between disease and all factors. Size was shown to be a consistent risk factor as turtles of around 75cm were most affected; a correlation was apparent between land use and disease according to nitrogen levels; and it was also linked to macroalgae. Furthermore, local spatial scales were found to be the most suitable for investigation of fibropapillomatosis as it varied locally, suggesting local causations ought to be looking in to. According to the paper, it generally seems as though human impacts may increase disease rates, as agriculturally dominant areas of land had high nitrogen footprints and also high disease rates. These high nitrogen levels also corresponded to increased disease as they encouraged the growth of non-native algal species which uptake a lot of the nitrogen. As this is passed on to the turtles during feeding and digestion, it may become available to the herpesvirus and promote infection.


Although the paper is quite frankly a little confusing, I think it is important that it has attempted to link several factors together in order to understand Green turtle fibropapillomatosis as ecological processes are complex and diverse and do not just occur between the host and its pathogen. Moreover, it is also interesting that human ecological impacts are considered instead of focusing purely on the marine aspect, especially as in this case human activities seem to be the main promoter of infection. Having said this however, I think the authors have been a little short sighted as many other factors could influence disease rates, especially in terms of nitrogen as oceanic cycling may unfold more of the story.


A review of: Van Houtan KS, Hargrove SK, Balazs GH (2010) Land Use, Macroalgae, and a Tumor-Forming Disease in Marine Turtles. PLoS ONE 5(9): e12900. doi:10.1371/journal.pone.0012900

Fibriopapillomatosis in Hawaiian Green Sea Turtles

Review of: Chaloupka, M. Balazs, G. H. Work, T. M. (2009) Rise and Fall over 26 years of a Marine Epizootic in Hawaiian Green Sea Turtles. Journal of Wildlife Diseases, 45 (4) 1138-1142.

The long lived and late maturing Green Sea Turtles have been present in our marine environment since the Dinosaurs. Over the years due to exploitation, green turtle numbers have become depleted, leading to their endangered status. Recently the emergence of a pandemic disease, Fibriopapillomatosis, which is associated with the presence of herpesvirus, has been affecting green turtle populations Worldwide. Fibriopapillomatosis (FP) is a disease similar to cancer, leading to tumour growth on the skin of the organism. The prevalence of this disease has increased in green turtle populations in Australia, Indonesia and the US over the past 2-3 decades. This disease emergence is thought to have impaired the already depleted green turtle numbers, especially in Hawaii where FP is a major cause of green turtle stranding.

This study was the first long-term assessment of FP prevalence in any marine turtle population, and has been recording information on FP in green turtles in Palaau, (Molokai, Hawaii) since 1982. The population in Palaau has the highest recorded numbers of FP in the Hawaiian archipelago where the disease is endemic. A programme was set up to annually monitor FP in green turtles by capturing, tagging and recapturing individuals. These turtle were evaluated for FP and given a severity score ranging from 0-3. Data collected over a 26 year sampling period (1982-2007), allowed for estimations of FP prevalence based on the proportion of green turtles with FP at each annual sampling. The data indicated that prevalence of FP increased following the 1980’s outbreak, peaked during the mid 1990’s and has steadily declined since. Observations have also shown that not all diseased turtles die; many have been shown to completely recover from the disease.

In 1978 the green turtle was protected under the US Endangered Species Act, due to its declining numbers; although it has been suggested that the presence of FP will affect recovering stock numbers, green turtle numbers have been shown to be ever increasing. The causes of FP are unknown, along with the role of the alphaherpesvirus in disease causation. However, disturbingly it has been shown that green turtles only contract the disease on entry to coastal developmental habitats, suggesting that the cause for disease is in near shore foraging habitats.

The authors have proposed two plausible explanations for the prevalence of FP. Firstly they suggest that the decrease in turtles infected with FP could be as a result of herd immunity to the infectious tumorigenic agent (if herpesvirus is contributing to disease) and secondly the removal of tumour-inducing agents in the near shore foraging habitats around Molokai. Both explanations seem possible and when compared to FP prevalence in Florida it was mentioned that the population was stable, unlike the decrease in prevalence in Hawaii. This could be an indication that the genetically isolated Hawaiian turtles may have evolved immunity. I believe it is highly possible that the herpesvirus is the tumour causing agent present in the near shore foraging habitats, which the isolated turtle population have managed to develop resistance against.

Herpesvirus now endemic in Australia

A review of: Whittington, Crockford, Jordan and Jones. Herpesvirus that caused epizootic mortality in 1995 and 1998 in pilchard, Sardinops sagax neopilchardus (Syeindachner), in Australia is now endemic. Journal of Fish Diseases. 2008. 31, 97-105.
In 1995, a massive epizootic occurred in pilchards, starting in Australian waters and spreading bidirectionally over 6000km, throughout a range of species, and finally reaching to New Zealand. The disease was characterized by severe branchitis and was attributed to a herpesvirus, Pilchard herpesvirus (PHV). Mortality was high with vast numbers of dead fish washing onto beaches and settling on the seafloor also. Unsurprisingly, there were measurable secondary impacts in piscivorous species including penguins, which experienced increased mortality rates and reproductive failure due to food shortage.
The cause of the outbreaks of PHV is unknown although it is hypothesized that the virus was introduced to a naïve population, or due to the reactivation of latent infection within the population. The authors set out to determine whether PHV is still present within Australian pilchard populations as previous surveys were hampered by the fish’s wide geographical distribution and effective capture avoidance behavior. Observations and modeling of the spread of PVH disease in the 1995/1998-99 epizootics suggested a single semi-continuous Australian pilchard population with direct contact between shoals of adult fish and between schools within shoals, sufficient to promote the spread of disease. However, preliminary survey results from this experiment suggest that there are 4 separate populations now present in the waters around Australia.
Samples of fish were taken randomly from each sup-population and tested for PHV. The results clearly show that three of the four subpopulations were infected. The fourth subpopulation was not sampled using the same random technique as the other three, and so true prevalence is not certain. The authors conclude that their results show PHV is endemic in S. sagax neopilchardus in Australian waters, supporting the hypothesis that the virus was introduced to a naïve population in 1995 and again in 1998 when it became established. Those fish are now all dead either due to infection or lifespan. The PHV positive fish sampled in this study were therefore recently exposed to the virus and may be immune.
The findings of this study have implications for fisheries managers. If similair other surveys are carried out in the future with comparable results, it may be beneficial to manage anthropogenic influences to limit contact between infected and non-infected populations. Current international translocations of baitfish, including pilchards, clearly place naïve populations at risk of acquiring pathogens to which there is no prior exposure or immunity. Not to mention economic losses associated with infections in aquaculture and fisheries, there are potentially larger consequences as food chains are impacted and predator prey relationships change.
This paper was a bit long-winded although the authors do highlight potential problems and unreliability of their data, especially in regard to the 4th uninfected/ immune subpopulation. I think it would be very beneficial for more surveys like this to be carried out on a wider range of species; especially those used extensively in the production of fish meal for use in aquaculture, as many diseases can be contracted form the ingestion of previously infected food. It may help prevent some of the more important diseases, such as VNN, from having such a significant presence in aquaculture stocks.



Monday 30 January 2012

The Pervasiveness of Domoic Acid


Domoic Acid (DA), is a diatom-produced neurotoxin responsible for a severe neurologic and gastrointestinal illness called Amnesic Shellfish Poisoning (ASP). Planktivorous organisms have been identified as the vectors of the toxin and since 1987 several DA poisoning and mass mortalities of sea birds and sea lions have occurred worldwide. Although no confirmed DA toxicity events have been reported in whales, this study shows that humpback and blue whales can be exposed to the toxin through the consumption of DA contaminated prey such as krill and planktivorous fish.

In this study, anchovies and sardines viscera, as well as humpback (Megaptera novaeangliae) and blue whale (Balaenoptera musculus) fecal samples, have been analyzed with HPLC-UV methods for the presence of DA. All whale prey and fecal samples collected during August and September in Monterey Bay (CA), were found to contain DA at levels ranging from 75 to 444 μg/g in fish viscera and from 10 to 207 μg/g in whale feces. The collection dates corresponded to a bloom of the DA-producing diatom Pseudo-nitzschia australis and whale prey and fecal samples where found to contain DA only when diatoms were detected in surface waters at densities ≥103 cells/l. In addition, scanning electron microscopy (SEM), revealed the presence in the whale feces of numerous fragmented P. australis frustules, confirming in this way that whales were exposed to DA during the diatom bloom. Since krill break apart the diatoms on which they feed, the frustule's fragments were likely derived from ingested krill gut rather than directly from the water around the whales.

Using known DA levels in prey and the average feeding rate of humpbacks, the authors estimated a daily oral dose of 1.1 mg DA kg−1 for an humpback whale feeding on contaminated fish. Blue whales instead, feed exclusively on krill (Euphausia pacifica) and thus the oral dose received by a whale feeding on contaminated krill was estimated by the authors to be 0.62 mg/kg. For these calculations, resting metabolic rate was used, but clearly metabolic demands are higher when growth, movement, and reproduction are considered. In addition, during particularly toxic or dense blooms, fish and krill could contain higher toxin levels resulting in higher oral doses. It is still not known whether these doses are sufficient to induce neurotoxicity in whales, but it has been suggested that during dives marine mammals may become more sensitive to toxins due to their ability to shunt blood to vital organs (heart and brain), while decreasing circulation to organs involved in detoxification (liver and kidneys). In this case, the estimated oral doses can become theoretically sufficient to induce neurotoxicity in whales.

In addition to whales, the authors found that also fish from both benthic and pelagic communities were noticeably exposed to DA. Fish as diverse as Pacific sanddab (Citharichthys sardidus), chub mackerel (Scomber japonicus), albacore (Thunnas alalunga), petrale sole (Eopsetta jordani), jack smelt (Atherinopsis californiensis) and walleye surfperch (Hyperprosopon argenteum) were found to contain DA at levels ranging from 1.4 to 275 μg/g viscera during the toxic Pseudo-nitzschia bloom. Sanddabs, which are bottom feeders, presumably consume toxin that has reached the sea-floor via the sinking of toxic microalgae, while albacore and mackerel, which are pelagic feeders, commonly feed on krill and anchovies, an obvious source of DA during toxic blooms.

Concluding, DA may be rapidly transferred throughout the whole marine food web, from algae to whales and to many other components of both pelagic and benthic ecosystems making them potential victims of DA toxicity. During toxic blooms, planktivorous krill, anchovies and sardines can accumulate DA providing a direct link between toxic algae and higher level consumers. As a result, whales may be negatively impacted by DA toxicity, if they can accumulate sufficient levels of toxin via consumption of toxic prey.  


Reference: 
Lefebvre KA, Bargu S, Kieckhefer T, Silver MW. (2002). From sanddabs to blue whales: the     pervasiveness of domoic acid. Toxicon 40:971–7.

Changing seasons changing communities: A preliminary Study

A review of: Pereira, C., Salvador, S., Arrojado, C., Silva, Y., Santos, A., Cunha, A., Gomes, N., Almeida, A.. (2011). Evaluating seasonal dynamics of bacterial communities in marine fish aquaculture: a preliminary study before applying phage therapy. Journal of Environmental Monitoring. 13 (4), 1053-1058.

The use of phage therapy presents a very promising alternative to the use of antibiotics and harmful chemicals to control pathogenic bacteria in aquaculture. Before phage therapy can be effectively used a comprehensive understanding of bacterial community structure and bacterial population density is necessary. This study aims to develop the understanding of how seasonal dynamics affects overall bacterial community structure, microbiological water quality and population dynamics of disease-causing bacteria. This information will allow them to identify the optimum time period that phage therapy should be applied. The study was carried out in Marine aquaculture system off of the Ria de Aveiro in Portugal. The aquaculture is located near the densely populated Aveiro and as a result the water is subjected to contamination by introduced human wastes. Currently as a countermeasure the water is selectively treated with chemicals, depending on the bacterial community structure, to maintain good water quality.



Samples were collected at different seasons in the year over 2 years. They measured temperature, salinity, dissolved oxygen on site and then within 1-2 hours they processed the samples in the lab. This provided detailed information on the seasonal changes of the water properties. They quantified the relative abundance of specific groups of bacteria using FISH analysis with 16S rRNA target probes. They evaluated the seasonal dynamics of bacterial community structure by comparing DGGE profiles of 16S rDNA fragments. They analysed bacterial indicators such as faecal coliforms and faecal enterococci by filtering and culturing samples. The results were expressed as colony forming units.



DGGE analysis showed that there was highest diversity of bacterial ribotypes occurred during the spring season. The quantification of bacterial indicators showed that in the coldest months (October, November and December) there was a decrease in the number of faecal coliforms, However the highest value of enterococci was obtained in October. The most dominant pathogenic bacteria found in the culture varied between seasons and years. Overall total bacterial numbers were fairly constant over the year but the relative abundance of specific bacterial groups varied significantly during the sampling period. There was a significant amount of variation community structure in the Vibrio genus. As Vibrio bacteria are known to be play an integral role in fish disease outbreaks it will be vital to consider these changes when phage therapy is applied. The effects of chemical disinfection also need to be taken into account as it has a serious effect of community structure. From this preliminary study they conclude that the most effective time to apply phage therapy in this system would be in the spring season.


It is interesting that the quantity of the faecal indictor enterococci was found to be highest in a time period that coliforms were lowest. I assume this is the result enterococci outcompeting coliforms because they are more adapted to thrive in the October conditions. The study has been effectively executed providing reliable information by taking triplicate samples and comparisons against controls. The high number of variables that would have had an impact on bacterial communities has been closely monitored and controlled in this study. This is a preliminary study which will hopefully lead onto the further research. I have had a look for this next paper but was unsuccessful finding it. I will keep looking and hopefully I will be able to review it and make it my next blog post.

Domoic Acid Production in Polar Regions

Domoic acid (DA) is a neurotoxin that is produced by phytoplankton and causes amnesic shellfish poisoning (ASP) in mammals, including humans. It may cause memory loss, brain damage or may even be fatal if consumed. DA is of concern to humans as it may accumulate in shellfish, which we consume, as they feed on phytoplankton that produce the toxin. Algal blooms of a species that produces the toxin are a good indicator that the toxin will have accumulated in the surrounding shellfish.


In this experiment single cells or colonies were isolated from samples of Pseudo-nitzschia seriata collected off the coast of Greenland. This paper investigates the effects of temperature on the production of DA in P.seriata, comparing the production of DA at 4oC to that at 15oC. The effects of temperature on the morphology of the frustule in the diatom were also tested. The density, morphology and layout of the poroids within the frustule were tested, comparing poroids at 4oC to those at 10oC and 15oC.


Before this study no diatoms had been found to produce DA in polar waters, although P.seriata had been found to produce the toxin this was found in samples taken from temperate waters. The findings of this study have however shown that P.seriata is able to produce DA at temperatures of 4oC and that the diatom didn’t produce any DA at 15oC, likely due to a decrease in cell number due to the temperature stress.


It was also found in this study that the number of rows of poroids, as well as their density, decreased in P.seriata with increasing temperatures. The morphology of P.seriata grown at 15oC was compared with species of diatoms that grow in temperate regions and the morphology was very similar, showing that using the morphology of the frustule in diatoms may not be a good way of differentiating species. Although there were a lot of similarities in morphology there was usually a distinguishing feature, such as a higher density of poroids in P.seriata compared with the temperate diatom P.australis.


This study highlights that the production of DA has been shown to increase with longer photoperiods in diatoms, which is why it is particularly important to investigate the production of DA in P.seriata from Polar Regions as during the summer these areas may be exposed to sunlight for long periods of time. A further study on the increase in DA production of P.seriata with increasing periods of light exposure would give a better idea of how big a problem arctic diatoms may be in the accumulation of DA in the arctic food web. The results of this study should be taken into consideration by authorities in arctic regions when they are monitoring the levels of toxic diatoms in the area as the current limit in many countries (before the levels are tested in plankton and mussels) of 200,000 toxic diatom cells L-1 is thought to be too high, which may be putting many people’s healths at risk.




Reference: Hansen, L et al. (2011), Toxin production and temperature-induced morphological variation of the diatom Pseudo-nitzschia seriata from the Arctic, Harmful Algae, Vol 10, pg 689-696

Sunday 29 January 2012

Bacteria make shellfish safe to eat!?


Paralytic shellfish toxins (PSTs) are produced by marine dinoflagellates, and pose a serious threat to the human food supply due to their high toxicity and lack of available medical treatment. PSTs concentrate largely in the digestive tract of bivalve molluscs, which filter feed on marine algae. These then pass into the human food chain through ingestion of the molluscs, which are not affected by commercial sterilisation or cooking. There are currently no practical methods for the detoxification of living shellfish.

The presences of toxin-transforming enzymes and/or microorganisms in bivalve molluscs have been suggested, due to several reports noting that their digestive glands have a high capacity for PST transformation. The discoveries of bacterial degradation of toxins suggest that bacteria may play an important role in the elimination of toxins from toxic bivalve molluscs.

It has recently been reported that bacteria found within blue mussels are capable of breaking down marine toxins more rapidly than those of other bivalve species. This report assesses the phenotypic and taxonomic characterisation of these bacteria.

Toxic blue mussels were collected from around Atlantic Canada, and microflora from their digestive glands was cultivated on agar. 69 bacterial isolates were identified and grouped according to the colony appearance. All isolates were then tested for the ability to break down PSTs, and cultures were incubated at 25oC for 5 days in a shaking incubator. Samples were taken on days 0 and 5, and were analysed for PSTs. It was found that only 7 isolates completely eliminated one type of PST, and reduced the overall toxin by no less than 90% in less than 3 days. These findings were confirmed by further study, which implies true biodegradation. Analysis of the 16S rRNA genes indicated that all 7 isolates were composed of bacteria belonging to a single clade within the genus Pseudalteromonas. These are often found in association with toxic dinoflagellates, and have been reported to produce a number of biologically active metabolites, including antibiotics and antimycotics. Some species within this genus have also shown algalcidal activities against dinoflagellates producing PSTs. Transmission electron microscopy revealed that all 7 isolates were rod-shaped and occurred as single cells and short chains. The isolates also possessed pili and flagella, and outer membrane vesicles.

In conclusion, it is possible that bacteria from within the genus Pseudalteromonas can assist bivalves in breaking down PSTs accumulated in their digestive tracts. Work is currently underway to support these findings, and to develop a biological process that will enable the elimination of these toxins from bivalves in vivo.

This report seems to be a preliminary study that has been used to support further investigation by the Canadian food inspection agency. Its findings in this early stage suggest the possibility of promising results in future studies such as this, and could ultimately lead to a higher level of control over the levels of PSTs present in marine Bivalves fit for human consumption.

A review of Donovan C.J., Garduno R.A., Kalmokoff M., Ku J.C., Quilliam M.A. and Gill T.A. (2009) Pseudoalteromonas Bacteria Are Capable of Degrading Paralytic Shellfish Toxins, Applied and Environmental Microbiology, 75, 6919-6923

Use of bacteriophages in fish aquaculture

A review of: Periera, C. et al (2011) Bacteriophages with Potential for Inactivation of Fish Pathogenic Bacteria: Survival, Host Specificity and Effect on Bacterial Community Structure, Marine Drugs, 9: 2236-2255.

The aquaculture industry is very fast-growing, with an average 6% increase in production per year reported over the last decade, but incidences of disease caused by pathogenic bacteria have led to great financial losses. Solutions to this problem have included the use of vaccinations and antibiotics, but these are not without their drawbacks. Vaccinations are of limited use owing to the range of different diseases present in a variety of fish species, and overuse of antibiotics has led to the emergence of multidrug resistant bacteria.

Previous studies have shown that the use of bacteriophages to control bacterial disease in fish aquaculture has great potential, but the success of phage therapy depends upon the ability of the viruses to survive and remain infective in the water, and their effects on non-pathogenic bacteria, which have an important role in aquaculture, particularly in semi-intensive systems.
The aim of the study was to isolate Aeromonas salmonicida phages (AS-1) and Vibrio parahaemolyticus phages (VP-1) and investigate their survival, specificity, and impact on natural bacterial communities within a semi-intensive system in Ria de Aveiro, on the North-Western coast of Portugal.

Analysis of the host range of the phages showed that AS-1 was also able to infect Vibrio anguillarum (98.87% efficacy) and V. parahemolyticus (96.03%), and VP-1 also infected V. anguillarum (83.27%) as well as A. salmonicida (64.75%).

Survival of the phages was determined using quantification through the soft agar overlay technique. AS-1 phage had a survival period of 91 days, whereas the survival period of VP-1 was much lower, at 16 days.

Analysis of DGGE profiles of PCR-amplified 16s rRNA gene fragments showed that bacterial ribotype diversity was not significantly altered by the addition of the phages to aquaculture water samples, after ten hours of incubation.

As fish aquaculture continues to grow and concerns about the widespread use of antibiotics within the industry increase, it is important that viable alternatives are found to control disease caused by pathogenic bacteria. The two bacteriophages isolated in this study show long term viability and an ability to infect a range of hosts without having a detrimental effect on natural bacterial communities. The results of the study suggest that more research should be conducted in order to devise an effective combination of phages for use in phage therapy. The authors also point out that owing to the seasonal variation within bacterial communities, careful monitoring should take place so suitable phages are selected.

Panaeus monodon and their immune- related genes.

A review of: Soonthornchai, W, et al. 2009. Experession of immune- related genes in the digestive organ of shrimp, Panaeus monodon after an oral infection by Vibrio harveyi.

Production of the cultivated shrimp Panaeus monodon has increased massively over the past 3 decades. This rapid increase has seen an increasing problem with disease outbreaks. Vibrios is the most common bacterial disease that cause mass moralities of shrimp world wide. Shrimp are exposed constantly to a variety of bacteria and viruses, and hence the defence mechanisms they employ.
Most of the immune response knowledge known to date has been obtained through the direct injection of bacteria into the body cavity or tissues of the organism. this approach has shown to be effective in identifying host mechanisms. But it bypasses the natural entry route of infections and the subsequent paths within the organism. This study instead used an immersion technique. This is a more natural technique of simulating an infection.

Panaeus monodon juveniles (3-4 Months) were obtained from Thailand and an immersion technique was conducted using Vibrio harveyi 1526 (vh). Control shrimp were also used during the course of this experiment. RNA isolation of each sample was conducted. RNA was extracted using Trireagent. Reverse transcription PCR was then conducted, and the immune related genes were isolated. To confirm changes in the expression of immune-related genes such as penaidin, crustin and C-lectin, quantitative real time PCR analysis was conducted. The six genes that showed the biggest effect after bacterial infection were selected to determine the tissue distribution. Tissue samples of the digestive system were studied for immune gene expression following bacterial infection. All samples were then fixed in R-F fixative. paraffin embedded tissues were sectioned, stained and analyses under a light microscopy.
During the experiment fifteen immune-related genes were discovered, throughout the entire experiment all of these genes continued to be expressed in high levels. Only six of these genes were known to be effected by the bacterial infection.VH uses sophisticated strategies to counteract immune responses in the gut, their persistence usually results in colonization and rapid multiplication in the shrimps gut. The epithelium is damaged as the bacteria attempt to gain entry into the immune system. The natural method of simulating an infection used in this study proved useful in establishing the shrimps natural genetic defences against VH. In previous studies where direct injection of the bacteria was used the gene expression did not increase drastically in comparison to the heightened expression of genes using the immersion technique.

Friday 27 January 2012

Tetradotoxin producing bacteria?

A review of the paper: Wang, J., Fan, Y., Yao, Z., (2010), Isolation of a Lysinibacillus fusiformis strain with tetrodotoxin-producing ability from puffer fish Fugu obscurus and the characterization of this strain, Toxicon, 56, 640–643

Tetradotoxin (TTX) is a neurotoxin that can cause paralysis and death in humans. It was originally thought to be exclusively found in puffer fish although now it is known to be found in a wide range of taxa, both terrestrial and marine. It has recently been discovered that TTX productivity occurs in isolated forms of bacteria. The aim of this paper was to discover whether this productivity would be found in isolated bacteria associated with puffer fish.

Six female puffer fish (Fugu obscurus) were collected from China and transported to a US lab. The ovary, liver, intestine and skin were separated and the toxicity of each organ was analysed using a bioassay. The liver was also used for bacteriological examination. Bacteria were isolated, purified and cultured, and the toxin was extracted from the cultures. The toxicities of the extracts were also determined by an assay.

It was found that different organs contain different levels of TTX: the ovaries and liver had the highest, followed by the intestine and then the skin. Six bacteria cultures were found within the liver, one of which was a TTX producer (labelled B-1). The toxin extracted from this culture was injected into mice, which showed the typical symptoms of TTX poisoning. This bacterial strain showed a 98.4% genetic homology with the bacteria Lysinibacillus fusiformis. It is supposed that if there is more than a 98% homology, the two strains are of the same species. Therefore B-1 was determined to be L. fusiformis.

The amount of TTX produced by the bacteria was very low to account for the high levels of TTX within organisms. The authors suggested that this may be due to bioaccumulation within the animal but were still unsure. However, this paper did show that a bacteria associated with puffer fish was a producer of TTX, but more work is needed to determine the mechanism behind the accumulation of the toxin within animals.

Wednesday 25 January 2012

Antibiotic susceptibilities in fish pathogens

There is currently a shortage in the literature on the antibiotic susceptibilities of fish pathogens. In this review I cover a paper whose authors compare three different methods in the hope of examining and evaluating antibacterial susceptibility; (i) the broth microdilution method (ii) the Etest and (iii) the disk-diffusion method
Francisella noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis are small pathogenic bacteria with capabilities of causing piscine francisellosis (an acute to chronic disease) across a wide geographical range of different fish species. 10 bacterial isolates were obtained from two different fish species from four geographic regions between 2006 and 2010 (Escherichia coli ATCC 25922 was used as a control). To begin, the genetic homogeneity of all the isolates and the control were tested via PCR mediated genomic fingerprinting. Electrophoretic profiles established great levels of homogeneity between all 10 isolates and a significant difference from the E. coli ATCC 25922 control. It was concluded the isolates from different fish and different regions shared the same genotype.
When Soto et al. evaluated the use of the broth microdilution method, they found all the minimum inhibitory concentrations for substances (which reference values at 28°C are available) fell within the range given by the Clinical and Laboratory Standard Institute (CLSI), indicating this method is suitable way to gage the antimicrobial susceptibility of the of the two above mentioned bacteria and indeed any fastidious organism.
When the authors evaluated the use of the Etest, they first appraised Cystine Heart Agar supplemented with bovine haemoglobin (CHAH) as a potential media and found that it was indeed suitable. The MICs were very similar to the broth microdilution method, and although there isn’t currently any publication for testing bacteria isolated from aquatic animals, the Etest appears to be a suitable method for determining antibiotic susceptibility. It is pointed out, however, that further exploration surrounding this method is necessary.
As with the Etest, when it came to evaluating the use of the disk-diffusion method, CHAH was again considered as a potential media. Clear zones of inhibition were observed for the quality control in the CHAH for numerous antibiotics, but when these zone diameters were compared to those observed on Mueller–Hinton agar supplemented with 5% sheep blood (MHB) and the ranges given by the CLSI, only the zones from a few of the tested antibiotics fell within the ranges. Although not what was expected, the results from the disk-fusion method were consistent and repeatable and together with the low MICs for these antibiotics it is reasonable to conclude the bacterial isolates are intermediately/highly susceptible to these few antibiotics (florfenicol, tetracyclin, nitrofurantoin, gentamicin and erythromycin).
The paper concludes by giving a long list of antibiotics the bacterial isolates are susceptible too (18 in total), and also a long list of antibiotics that they are resistant too (26 in total).

This paper provides a good baseline of data for future research monitoring the development of antibiotic resistance among the bacterial isolates, as well as being a starting point for the development of potential therapeutics to bacterial diseases. The article was well structured and not overly complex to read and understand. Although I didn’t feel like I’d learnt much after reading it, I don’t feel that was the aim of the authors, the aim was to provide a very good and essential platform for further research.


A review of:
Soto, E., Griffin, M., Judy, W. and Hawke, J. P. (2012) Genetic analysis and antimicrobial susceptibility of Francisella noatunensis subsp. orientalis (syn. F. asiatica) isolates from fish. Veterinary Microbiology 154 407-412

Tuesday 24 January 2012

Watch out bacterial pathogens!

A review of: Selvin, J., Manilal, A., Sujith, S., Kiran. G.S., Lipton, A.P. (2011). Efficacy of marine green alga Ulva fasciata extract on the management of. Latin American Journal of Aquatic Research. 39 (2), 197-204

11 species of Vibrio are commonly recognised as members of the normal bacterial flora of shrimp culture systems. However some of these members such as V. alginolyticus and V. harveyi pose serious threats as opportunistic pathogens and have been known to have a serious impact on the shrimp production industry. Marine secondary metabolites, from a number of different sources, have been recognised in previous studies as promising agents in shrimp disease management. The marine algae Ulva fasciata is found on the southwest coast of India amongst diverse variety of seaweed species. This highly competitive environment would insure that this alga’s secondary metabolites are highly reactive. The aim of this study was to establish the effect and the efficacy of U. fasciata based feed on the survival of shrimps experimentally infected with a range of pathogens.
They initially found the found the Lethal dose (LD) value of the pathogens: V. fischeri, V. alginolyticus, V. harveyi and Aeromonas sp. They intramuscularly injected samples of shrimp with the pathogens and then observed the effects. They increased the dose until total mortality was observed. A control group was injected with saline solution. They then top-coated commercially formulated shrimp feed with varying quantities of U. fasciata extracts 500mg, 1000mg and 1500mg/ kg of shrimp. Different groups shrimp were fed with U. fasciata diet for 15 days prior to being injected with the LD of the different pathogens. Again they used a control group which was not given U. fasciata in their diet to compare results. The diets were continued for 15 days after they were inoculated with the pathogens and a number of physiological and behaviour features were observed.
Shrimp fed with 500mg had the lowest protection against bacterial infection. Shrimp fed with 1000mg showed the highest level of protection in comparison to the other doses. They found that the survival of treated shrimp against the bacterial infection was significant at P<0.01. They found 100% mortality in the control shrimps even before the lethal dose, found in the preliminary experiments, was met. They believed this could be due to enhanced virulence obtained by the pathogenic isolates passed through the host, prior to in captivity control experiments. The median lethal dose of algal extract was determined to be 1120mg which is most likely the reason why there is a higher percentage of infection in the group of shrimps treated with 1500mg of U. fasciata. In an earlier study it was observed that pathogens were cleared off from the shrimp’s haemolymph within an hour in groups treated with U. fasciata. This suggests that the secondary metabolites of the algae stimulate the rapid response of bactericidins inside the shrimps. This study presents a promising development in bacterial pathogen management using natural marine products. The extracts can be stored in a dry form for long periods and this coupled with how easy it is to extract the bioactive ingredients makes it a low cost solution. Algae can be cultured in large quantities making its secondary metabolites not only eco-friendly but sustainable too. The large number of similar studies recently published is not surprising considering that this multimillion pound industry can be so easily threaten by things that are so hard to see.

Monday 23 January 2012

Specificity, my dear Watson

The main objective of this paper is to try to understand why certain species of pathogenic bacteria can be species specific. Filter feeding bivalves process a lot of water and because of this they are exposed to a lot of bacteria. Most of these bacteria are degraded (in Mytilus edulis rates of up to 10⁹ cells an hour) however certain Vibrio species have been shown to inhibit this degradation. Bivalves’ first line of defence against pathogens is through the use of haemocytes, which from what I gather are kind of their version of leucocytes. Previous studies had shown that pathogens induced greater rounding of haemocytes in bivalves of which they are known pathogens than species of which they are non-pathogenic. This study tries to see if the interaction between pathogens and haemocytes is possibly the reason for certain pathogens specificity.

In this experiment they wanted to see the degree of rounding nine species of vibrios had on seven species of bivalves. Haemocyte monolayers were prepared by adding haemolymph from the bivalve samples to 24-welled flat bottomed microtitre plates. Haemocytes were given time to adhere and then any surplus haemocytes were removed. Washed vibrios were then added to the monolayer at a concentration of 50 bacteria per haemocyte. They were then incubated for three hours at 20°C. The rounded cells were counted through the use of photographs.

Overall there was some degree of specificity. M.edulis was the most sensitive species with six of the nine vibrios rounding at least 80% of the haemocytes. Two species of Vibrio were highly specific for individual species of bivalves with another Vibrio species which only affected the clam species.

They conclude that the most likely reason for this specificity is probably due to the receptors present on the haemocytes. It’s the most exciting or inventive conclusion but makes sense as there have been many recent papers which have shown toxins to be specific to certain phagocytic cells. Overall I thought this was a very interesting and well written paper with the added bonus of only being four pages long. What I find particularly interesting is that the main bacteria which cause this rounding are vibrios which are also the main marine human pathogens. Papers have shown that many of the adaptations which vibrios possess allowing them to infect humans have evolved in their natural environment for other reasons. Possibly they use this adaptation to defend themselves from haemocytes when infecting humans. This could explain why immunocompromised individuals are more susceptible to vibrio-related illnesses.

A review of: Lane, E. and Birkbeck, T. (2000) Species specificity of some bacterial pathogens of bivalve molluscs is correlated with their interaction with bivalve haemocytes. Journal of Fish Diseases 23: 275–279.

Antibiotics: more of a hinderance than a help!

A Review of: Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and the environment. F.C Cobello. Environmental Microbiology. 2006 8(7) 1137-1144.

Industrial aquaculture is a rapidly growing industry in almost every region of the world. Depletion of fisheries and the market forces that globalize sources of food supply are expected to stimulate an even faster growth rate of the aquaculture industry in the future. However this impressive industrial development has been accompanied by some practices potentially damaging to human and animal health that include passing large amounts of Vetinary drugs into the environment, resulting in an increase of antibiotic resistant bacteria and fish pathogens. Effective use of prophylactics in aquaculture is therefore undermined and increases the possibility of transferring this resistance to bacteria (including pathogens) of terrestrial animals and humans, proof of which has recently been shown in a number of studies.
Antibiotics are administered to fish as a component of their food and occasionally by injection or baths. Unconsumed food and faeces containing antibiotics reaches the sediment at the bottom of the fish enclosure where the antibiotics are leached from the food and faeces and diffuse into the sediment where they can be washed away by currents to distant sites and potentially ingested by wild fish and other organisms. Antibiotics that are not washed away exert selective pressure on the microflora of the sediment, altering the composition and selecting for antibiotic resistant bacteria. The author describes the results of many studies supporting the concept that antibiotic usage in aquaculture will influence the appearance of resistance in bacteria of other niches, including resistance to pathogens able to produce a variety of animal and human diseases.

Residual antibiotics in commercialized fish and shellfish products are another problem created by extensive antibiotic use in aquaculture. If undetected, potential alteration of the consumer’s natural flora may occur, increasing their susceptibility to bacterial infections and also selecting for antibiotic resistant bacteria. Problems related to allergies and toxicity may also occur.
As a result of the many studies into the effect and potential results of excessive antibiotic administration in aquaculture, many countries around the world have implemented drastic restrictions on antibiotic use and attention is now turning to the use of vaccines to cure and prevent many of the bacterial diseases commonly contracted by fish in aquaculture. However some countries, such as Chile and China have increased antibiotic use in recent years, form 30 tonnes to close to 100 tonnes as antibiotic use is still totally unrestricted.
This review is rather short but does bring together and summarize a large amount of research into the effects of antibiotic use in aquaculture with particular emphasis on wild microbial populations and potential effects on humans. However, it does not go into detail about how antibiotic consumption can have negative impacts on cultured fish and shellfish by ultimately decreasing the effectiveness of their immune system and therefore making them more susceptible to infections.

Friday 20 January 2012

Do Toxins Affect Aggression in Rainbow Trout?

A review of the paper: Bakke, M.J., Hustoft, H.K., Horsberg, T.E., (2010), Subclinical effects of saxitoxin and domoic acid on aggressive behaviour and monoaminergic turnover in rainbow trout (Oncorhynchus mykiss), Aquatic toxicology, 99, (1), 1-9

Algae in the Alexandium family produce saxitoxin and other analogues that are known as Paralytic Shellfish Posioning (PSPs) toxins, which affect the sodium channels in the nervous system. Another algal family that produce toxins are the Pseudonitzschia family, and these produce Domoic acid. This affects receptors in the brain, causing a suite of neurological symptoms. Both of these toxins have caused huge economic losses in the fish farming industry by causing mortalities and decreased growth via stress. They affect the brain of fish, so behavioural abnormalities should be expected. An important species of fish involved in farming is the Rainbow trout. This fish displays aggressive behaviour towards intruder fish, and protect their “home tank”. This aggressive behaviour is controlled by the serotonergic and domaminergic systems, which both of these toxins have been shown to affect in rats. The aim of this paper was to see whether these toxins affect the aggressive behaviour in Rainbow trout, and therefore if the toxins affect these systems.

4 groups of 8 Rainbow trout were placed into tanks and were exposed to intruder fish on 2 consecutive days. The intruder fish were always smaller than the resident fish to encourage aggressive behaviour. The first day was a control where no toxin was injected. On the second day, 4 different treatments were implicated: a control with no injection, a saline injection, a saxitoxin injection and a Domoic acid injection. Each fish was video-taped and the behaviour was analysed for: the time (latency) until the first attack, the number of attacks in the following 30 minutes and the type of attack (approach, chase or bite). If there was no attack in the first 15 minutes, the test was aborted. After the tests were complete, the resident fish were taken for tissue samples.

No significant differences were found from the behaviour of day one, however on day two there was a difference between the controls and the saline-injected fish, and the saline and Domoic acid-injected fish. The fish that were injected with the saline solution, showed a decrease in aggression. As there was no toxin in this injection, it can be deduced that this is down to the stress of handling. This suppression of aggression was absent in both the saxitoxin and Domoic acid groups of fish, despite the same handling stress showing that the toxins elevated activity. Seratonin levels were elevated all 3 groups of injected fish, showing elevated stress levels. The toxins did not affect the levels of monoamine or serotonin in comparison to the saline-injected group. However, this paper did conclude that the toxins masked the effect of the handling stress. To me, this implies that if the toxins were ingested naturally, there would be an elevated amount of aggressive behaviour but this paper has not been able to prove this.

Thursday 19 January 2012

Adaptation to Life in Ice

It is well documented that microbes can survive and even thrive in extreme heat. On the other end of this spectrum are organisms that live in extremely cold environments. During winter months in the Polar Regions large areas of ocean become covered by sea ice as sea water freezes. The temperature in this ice ranges between -1.8oC at the sea-ice interface and >-20oC at the air-ice interface. As the ice forms, salt separates from the water and small brine channels are created that act as drainage through the ice. Despite the extreme cold and high salinity, the brine channels are home to many micro-organisms. An important adaptation these organisms possess is the ability to produce a form of antifreeze. Different names for these molecules exist depending on the authors, but these include antifreeze proteins, ice binding proteins or ice structuring proteins.

One of the most dominant microbes in the Antarctic sea ice belongs to the diatom genus Fragilariopsis . In a study by Krell et al (2008) a species belonging to this genus called F. cylindrus was one of the first discoveries of a diatom to produce antifreeze proteins (AFP). Its discovery was actually an accident as they were originally examining this species ability to with stand salt stress. An increase in salt concentration caused the up regulation of four genes that encode for antifreeze proteins. Three of these genes were almost identical and the proteins they produce are believed to secrete from the cell and prevent the formation of ice crystals around them.

Bayer-Giraldi et al (2010) continued research on this topic to see whether more AFPs could be identified in F. cylindrus and another species, F. curta. Using PCR amplifications with different primers they were able to isolate the genes identified by Krell et al (2008) plus some additions. In total there were 10 in F. cylindrus and 11 in F. curta making up a multigene family in each species. Majority of the genes had very little variation between them and sometimes only resulted in a single amino acid replacement but as the protein structures have not been studied in detail, it is unknown if this has any relevance. The authors suggested that having multiple genes that are essentially the same would compensate for reduced kinetic activity within cells and their components. In other words, these cells can continue to produce AFPs in relatively high amounts even when enzyme activity has slowed right down as a result of low temperature.

A second objective of this study was to compare these genes with other groups of organisms. Fragilariopsis AFPs appear to be significantly different to other diatom AFPs suggesting that they were acquired through horizontal gene transfer from different microorganisms. Similarities with other microbes indicate Fragilariopsis acquired AFPs from bacteria or fungi but archaea were possibly the source for other diatoms. There were also remarkable similarities between some of the microbe AFPs and AFPs found in a species of copepod called Tephos longipes which also lives in Antarctica. This indicates that AFP genes are highly mobile and seem to play a very important role in polar adaptations.

A Review of:
Bayer-Giraldi M, Uhlig C, John U, Mock T and Valentin K (2010) Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis. Environmental Microbiology. 12: 1041-1052.

Additional Reference:
Krell A, Beszteri B, Dieckmann G, Glockner G, Valentin K and Mock T (2008) A new class of ice-binding proteins discovered in a salt-stress-induced cDNA library of the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae). European Journal of Phycology. 43: 423-433.