Survival of the Fittest

Aeromonas salmonicida (A. salmonicida) is a fresh water organism that affects cold-blooded animals such as fish. This gram-negative bacterium is known to cause furunculosis, a disease that is fatal to salmonid fish. Vibrio anguillarum (V. anguillarum) is also a gram-negative bacterium that affects fish and other aquatic animals such as eels. This very infectious bacterium causes a disease known as vibrosis. Treatments are available for both diseases but have little effect. Salmon farming is a huge industry in Canada and therefore research into fish disease is important to the stability of our economy.

This experiment tested the survival of A. salmonicida and V. anguillarum on two different types of aquaculture material; net pen and bag pen. In addition, the effectiveness of bleach and iodine to eliminate the bacteria from the material was tested.

Ten 50mL test tubes were filled with 25mL of filtered seawater for each of six conditions. Two 3cm pieces of nylon net, each containing a not, or 2cm² piece of bag were added to the appropriate tubes. A specific amount of colony forming units of each bacterium was added to the correct tubes. Two samples were taken from each condition at weekly intervals and assayed for the number of A. salmonicida or V. anguillarum colonies. In addition, samples from each condition containing bacteria were sterilized by two different methods: iodine and bleach.

From the results it was determined that bag pens, which are currently only used on the West coast of Canada, paired with an effective sterilization technique would provide the safest environment for salmonid fish from furunculosis or vibrosis. As salmon farming is such a large industry in Canada, an outbreak of furunculosis or vibrosis would potentially cause a decline in our economy. If we were to continue experimentation we would use a longer testing period and perform a larger number of dilutions in order to obtain more accurate results. In this experiment, because of the time frame, we chose two more common bacteria for testing. However, if given the chance we would use a larger range of bacteria to increase the relevance of the project.

In our study we determined if the bacterial species, A. salmonicida and V. anguillarum, can colonize materials used by the aquaculture industry. We tested the bacteria’s ability to colonize on 2 types of fish pens: bags and nets. We tested the physical and chemical sterilization methods commonly used in the aquaculture industry and determined which is most effective.

Aeromonas salmonicida is a fresh water organism that affects cold-blooded animals such as fish. This gram-negative bacterium has been found to cause a systemic disease in salmonid fish called furunculosis. A furuncule is a boil, and the appearance of one or more boils on the skin is a classic sign of furunculosis. Furunculosis is a major cause of mortality in salmonid fish. However, it can be treated with antimicrobial therapy, administered as a food additive, or with a vaccine. Unfortunately current vaccines offer limited effectiveness and sudden outbreaks (epizootics) are common in farmed fish.

Vibrio anguillarum is a gram-negative bacterium that affects fish and other aquatic animals such as eels. V. anguillarum is widespread, but is particularly found in Japan and North America. This highly infectious bacterium causes the disease vibrosis. Signs of vibrosis include hemorrhaging and erythema both internally, in the organs, and externally, at the base of the fins, the vent and in or around the mouth. Vibrosis outbreaks can only be treated with an oral antibiotic. However, this has little affect since infected fish stop eating. Furthermore, resistance to antibiotics is becoming a problem. V. anguilluarum has already become resistant to the commonly used oxytracycline, and is beginning to become resistant to other antibiotics.

Bag pens are a new development that are used in Chile and on the west coast of Canada. Bag pens provide a more solid separation between the fish and the environment. Water is pumped into the bags through pipes, allowing the fish farmer to control the temperature and oxygen levels of the water as well as how much water is being put into the bags. By adjusting the pipe to a higher or lower level not only can the oxygen and temperature levels be controlled, but also the fish can be protected from the toxic algae blooms found on the surface of the water. Other benefits of bag pens are the ability for waste to be stored, the ability to farm a variety of fish and the ability to protect fish from predators.

Fishnets are the most commonly used fish pens in the aquaculture industry; they are the only kind of pen used on the east coast of Canada. Nets are significantly cheaper than the alternatives, however they do not have the same advantages. For instance nets give little protection from predators and expose the fish to toxins such as algae blooms. The temperature, oxygen levels and food consumption cannot be controlled in fishnets. Fishnets tend to provide a more natural environment for fish rather than an ideal environment, like bags.

Salmon farming is a major industry in Canada, especially in the Maritimes. Commercial fishing for salmon occurs on a large scale employing various types of nets to catch the fish on their way to their spawning ground. Domestic salmon were first raised in Nova Scotia in 1967. Currently there are approximately 30 companies in Canada operating more than 200 salmon farms on both coasts. Most raise domesticated Atlantic salmon (Salmo salar) and Rainbow trout (Oncorhynchus mykiss). The Maritimes account for slightly more than one third of Canada's domestic salmon output. Approximately 90% of the Maritime production of domesticated Atlantic salmon comes from the Passamaquoddy and Grand Manan areas in New Brunswick. The remaining 10% comes from Nova Scotia and Newfoundland. Salmon farming is the fastest growing sector in world aquaculture.

Bacterial Survival - A section of nylon net was cut into a number of 3cm pieces each containing a knot. Similarly, a piece of tarp (to simulate the bag pens used in fish farming) was separated into 2cm² sections. A test tube rack was set up for each condition (Table 1) containing ten 50mL test tubes. Each tube was filled with 25mL of filtered seawater. 2 pieces of tarp were added to each of the tubes in three of the conditions and 2 pieces of net were added to each of the tubes in the three remaining conditions. One bag and one net condition were used as sterile controls. 4.4μL of an A. salmonicida solution with an optical density (OD) for one were added to all the tubes into one bag and in one net condition. 1.87μL of a V. anguillarum solution with an OD1 were added to each of the tubes in the two remaining conditions. This means that each tube, except for those in the sterile conditions, received 2.5 x 10⁹ colony forming units of bacteria. This information is summarized below.

Condition #	Bacteria	Net or Bag pen
1	No added bacteria	Net
2	A. salmonicida	Net
3	V. anguillarum	Net
4	No added bacteria	Bag
5	A. salmonicida	Bag
6	V. anguillarum	Bag

In addition, 4 tubes were filled with 25mL of seawater and the aforementioned amount of A. salmonicida was added to 2 of the tubes and V. anguillarum to the others. These tubes were left without fish pen samples as to assay the bacteria living in the water alone.

Each week, for a total of 6 time points, one tube was taken from each condition for sampling. Under a sterile hood, the water was poured off every tube and each sample of bag or net was placed in its own plastic bag. 90μL of phosphate buffered saline (PBS) was added to the bags before they were placed in a stomacher machine to re-suspend the bacteria. 10μL were removed from each bag and each of the water sample tubes using a pipette. Ten fold dilutions were made in tubes containing 90μL of PBS. Each of the four most appropriate dilutions were plated in three 10μL drops on quartered petri dishes of Tryptic Soy Agar (TSA). However, conditions containing V. anguillarum were plated on TSA plus salt.

The plates were incubated at 17 degrees for 4 days. On the fourth day the bacterial colonies were counted and charted. Every week the most easily counted dilution of each condition was chosen. The average of the three drops was multiplied by the dilution factors as to find the number of colony forming units per sample.

Sterilization Techniques – Four 50mL test tubes were filled with 25mL of water. 500μL of bleach were added to two of the tubes and 250μL of iodine were added to the other two. One tube was removed from each of the conditions containing bacteria. The net and bag samples were placed in the solutions according to Table 2.

The samples were left for 15 minutes and then removed and rinsed with sterile seawater. The samples were each placed in a plastic bag and the bacteria was re-suspended in 90μL of PBS. 10 fold dilutions were made and plated in the same fashion as in the ‘Bacterial Survival’ section of the experiment. The plates were also incubated at 17 degrees for 4 days and then assayed for the amount of bacteria present.

Bacterial Survival - No counts were seen for any of the conditions in week one due to the bacteria’s need for time to adhere to the fish pen material. Conditions 1 and 4 contained no bacteria and no colonies were seen throughout the duration of the experiment (blue and aqua lines respectively, Figure 1). It was seen that the number of bacteria in condition 2 decreased gradually and by week five could not be detected in our dilutions (fuchsia line, Figure 1). The amount of bacteria in condition 3 increased steadily and dropped slightly before rapidly increasing in the final week (yellow line, Figure 1). The bacteria in condition 5 did not survive well as it fluctuated a fair amount but died off by week 5 (purple line, Figure 1). Condition 6’s bacteria did not survive for a long time since it steadily decreased throughout the experiment and was virtually undetectable by the final week (brown line, Figure 1). It was also seen that throughout the duration of the experiment a fair number of both types of bacteria were able to survive in the water. However the sample of A. salmonicida experienced a rapid decrease in the first week but maintained a steady number of counts for the rest of the experiment (navy line, Figure 2).

Sterilization Techniques - It was found that no colonies of any kind were present after the selected sterilization techniques were performed. Both a 1:50 concentration of bleach and a 1:100 concentration of iodine were effective in eliminating bacteria in a 15 minute period.

Summary – It was found that other than V. anguillarum on net (condition 3) the bacteria in the other conditions were very similar in their rates of fluctuation. In addition, the other conditions had, generally, very few counts by the last week of the experiment. Both bacteria, especially V. anguillarum, were able to colonize on the fish net samples due to the woven fashion of the material. It is possible that V. anguillarum is able to survive on nets easily due to the size of the bacterium. The large V. anguillarum colonies are more likely to catch in the mesh of the nylon than the small A. salmonicida colonies. On the other hand both bacteria were unable to easily colonize on the surface of bag pens possibly due to the absence of pockets on the smooth surface.

The results also showed that iodine and bleach are extremely effective techniques for sterilizing both bag and net pens.

Application - While A. salmonicida did not survive well on the nets V. anguillarum did. Since, it was observed that V. anguillarum thrived on fish net, we can therefore conclude that using nets in an area containing V. anguillarum can increase the risk of vibrosis. On the other hand bag pens did not provide an extremely desirable surface of colonization for either bacterium. Moreover, bag pens are more suitable to a fish-farming environment owing to their ability to resist the colonization of bacteria. Nets could provide a safe environment for fish if they are sterilized at regular intervals with specific concentrations of iodine or bleach. Ideally bag pens, which are currently only used on the West coast of Canada, paired with an effective sterilization technique would provide the safest environment for salmonid fish from furunculosis or vibrosis.

If we were to continue experimentation we would use a longer testing period and perform a larger number of dilutions in order to obtain more accurate results. In this experiment, because of the time frame, we chose two more common bacteria for testing. However, if given the chance we would use a larger range of bacteria to increase the relevance of the project.

Economic consequences – Because both A. salmonicida and V. anguillarum grow at a healthy rate it would not take much bacteria to infect a fish. Since both furunculosis and vibrosis are common fatal diseases in Canadian fish farms, the use of the most efficient pen material is important. Today, a 3kg fish markets at approximately $24. An epizootic of either of these diseases could potentially cost a farmer thousands of dollars.

We would like to acknowledge the following people from helping us with our project. Firstly we wish to thank the National Research Council of Canada – Institute for Marine Bioscience NRC-IMB and our mentors, Dr Jessica Boyd and Dr Andrew Dacanay. We also wish to acknowledge the faculty of Sacred Heart School of Halifax and our science teacher Mrs. Davinder Singh. We wish to thank our parents Mr, Mrs Miller and Mr Tufts, Mrs Amit. Finally we wish to thank Aventis Biotech Challenge Organizers for funding.

Bruno D, Poppe T; Bacterial Diseases in A Color Atlas of Salmonid Diseases (1996); Academic Press

Tube Number	Type of Solution	Type of sample
1	Iodine	Condition 2 and 5
2	Iodine	Condition 3 and 6
3	Bleach	Condition 2 and 5
4	Bleach	Condition 3 and 6

A Test of Bacterial Survival and Sterilization Techniques

Ainsley Miller and Julia Tufts

Condition #