FALMOUTH MARINE SCHOOL
UV Sterilization and its role in reducing a Costia infection UV Sterilization – Costia Infection Kimberley Majer 4/14/2011
species is teardrop shaped or pear shaped. This shape means that it has a groove on one side that leads to a cytostome and this is where the flagella, of the protozoan attaches. This species can reproduce both asexually and sexually, usually they reproduce asexually by binary fission which means they spread across the host a great deal faster, than they would if the species were to reproduce sexually, which only occurs when the conjugation happen amongst the species. This species can also leave tough nonreproductive cysts in the host which can be only seen under the microscope as they are only 7 to 10 microns in diameter (Post, G. 1987).
Abstract Costia is a flagellate parasite that is normally found in the gills and skin of many freshwater and marine species and with Cyprinus carpio being the most popular ornamental fish ,highly valued, this experiment is involving this species (Sommerville C. 1984). The protozoan can be fatal in high numbers so can cause losses in aquaculture around the world. The experiment was ran for 3 weeks to gather a significant about of data to do a study how UV effected how the parasite spreads and survives amongst the systems. Using statistical analysis the null hypothesis was accepted due to there not being a significant difference between the systems. The graphs however showed there was a difference with the UV being used on a system as the parasites did not spread to the non – infected fish and decreased in number over the time period. Whereas on the system which did not have a UV sterilizer the parasite increased in number on infected specimens and spread to the non-infected fish.
Costia are a mutualistic parasite when they are found in small numbers they do not seem to affect the host and live off the shedding epithelial cells and other cellular debris of the host. So if the general health of the host is poor the normal mortality of the parasite can get affected (Robertson D, A., 1985). This species can reproduce at a phenomenal rate so they can become harmful as they can start to attack the living cells of the host. The parasites are usually found in the skin and the gills of the host as they multiply symptoms become apparent. The symptoms are quite easily spotted as physiological stresses begin to occur such as excessive mucus production, which makes the host have a milky white appearance on the surface; this also affects their respiratory system as the mucus clogs up the gills of the fish. The fish then become lethargic they also begin flashing as their skin becomes irritated (Post, G. 1987). As well as this they exhibit clamped fins and the eyes of the fish can become sunken. If the fish is left untreated the parasite will multiply at a considerable rate which can lead to fish mortalities. So it is essential that the infected fish are detected and treated for Costia infestation can be treated by using a mix of Malachite Green and Formaldehyde also potassium permanganate can be used. UV sterilization can be used to help with the reduction of the amount of parasites that spread amongst infected fish and noninfected fish (Noja, E, J. 2010.). UV sterilization is the use of ultra violet light to break up free floating protozoans, bacteria and algae that is not wanted in the system. The sterilizer breaks down the cells by using UV light to alter the structure of the cell’s nuclear material this results in the organism not being
Keywords Protozoans, epithelial layer infestation, UV sterilization, Cyprinus carpio, symptoms.
Introduction There is a growing economical value of the aquaculture industry. It is vital that diseases that can cause the value of the fish can decrease in the stock, are diagnosed and dealt with. With Cyprinus carpio being one of the most sort after fish in the industry it is essential that the health and welfare of the fish are kept to a high standard so the value does not diminish at all (Weaver. D.E, 1991.). One of the diseases that affect this species is Ichtyobodiasis (Costiasis). The two species that are the origin and cause of this disease are Ichthyobodo necatrix and Ichthyobodo pyriformis. They are both quite small for mastigophoran protozoa, I. necatrix are 10 to 20 microns long and 5 to 10 microns wide and the smaller of the two I. pyriformis at 9 to 14 microns long and 5 to 8 microns wide. They have short rod like structures called axostyle which are close to the body of the parasite and help to with the rigidity of the protozoan’s body as well as this they help with feeding. Along with this they have flagella which are longer than the body and are used to propel the organism and it helps the organism to attach to the host. The two species consist of vacuole that is capable of contracting and have a round vesicular nucleus. The body of this 2
able to reproduce which inhibits the spread of the unwanted organism. UV sterilization is carried out by running the water from the system into a chamber which contains a Ultra violet light bulb that is contained a glass sleeve in the shape of a test tube. This also occurs when the water leaves the system as there is an outlet chamber. So an experiment will be conducted to see if UV sterilization has a significant effect on reducing the spread of a number of Costia parasites amongst a group of infected Cyprinus carpio and a group of non- infected Cyprinus carpio (Fig 1a-b).
was conducted every week so cross contamination didn’t occur. The fish were fed on fishmeal pellets once a day and were observed to make sure there were not any signs of distress if there were any signs of distress the experiment would be terminated. The water quality of the system was monitored on a daily basis using Tetra test kits. The levels are checked with a colour chart from the tetra test kits all the TAN, pH and nitrite were kept at the correct levels throughout the experiment. This was carried out because if the water quality was poor the fish’s health would be affected and the amount parasites would be affected as well. The tank temperature was kept at 20˚C. The tanks were set up with the filter and pump running for two weeks before the experiment started so that the filter had time to mature and build up the bacteria in the biological filter so that nitrogenous waste could be broken down (Crosby, D. 2001). The first of the infected fish were introduced into the tanks two in the tank with a UV Sterilizer and two with no UV Sterilizer running then two non-infected fish were introduced to each of the tanks. They were acclimatised to the system over 45 minutes slowly introducing system water into the bag every 5 minutes. Once all the fish were in each of the tanks the UV sterilizer was turned on and left for 24hours. Then observations were carried out over the next 72 hours (Fig 2a – 2e).
a)
b) Figure 1a) is a picture of the parasite Costia taken from (Post, G. 1987) Figure 1b) is a photo taken of the species Cyprinus carpio photo taken by (Majer, K. 2011)
Materials and methods Fish Maintenance Two groups of fish, consisting two non-infected C. carpio and two infected C. carpio were kept in two separate 1000 litres that were filled with 500 litres of freshwater that had a pressurized filter with a turnover rate of 300 litres. One of the tanks had a hang on UV sterilizer attached to the side of the tank it was a Vecton 400. As well as this there was a pump attached to the tank so that the system was aerated sufficiently. The tanks were set up for 3 weeks a complete water change
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a)
d)
c)
b)
e)
Figure 2a -e this is a series of photos of the materials used in fish maintenance all photos taken by (Majer, K. 2011). Figure 2a A pump aerating the system, Figure 2b This is the two infected fish being introduced to the system Figure 2c This is the UV Sterilizer used on one of the tanks Figure 2d This is a filter that keeps the TAN at a low level and Figure 2e This is one of the 1000 litre tanks used.
Sample collection The observations began 24hrs after the fish were placed in the tank with one tank having the UV sterilizer on and the other tank just being left as is. To observe how many parasites spread or multiply in each system regular skin scrapes were carried out every 24hrs on each fish, for three days this was repeated for 3 weeks.
To conduct a skin scrape first a tray was prepared by placing a moist paper towel on the trowel so that the fish would not get dehydrated. The fish was then removed from the system using a fine mesh net, making sure that the fish was kept as calm as possible.
b) c) a)
d) Figure 3a-d this is a series of photos of the method for sample collection Figure 3a the fish being removed from the tank, Figure 3b this is the fish being held in the tray while a scrape is being conducted, Figure 3c this is the sample being transferred to the slide and Figure 3d this is the slide being prepared
Then the fish were placed on the tray and held with gloved hands. Using a blunt scalpel a thin layer of mucus is removed from the epithelial layer. The fish is then placed back in the tank so that dehydration was minimised (Noja, E, J. 2010). Then the sample was placed on a slide with a drop of water to make sure the sample does not dry out. Once the slide was prepared it is placed under a microscope to begin the identification of the bacteria (Fig 3a-e). Microscopy and bacteria identification
H1 There is a significant difference UV sterilization having an effect on the spread and multiplication of parasites in a system.
The slide was then placed underneath a microscope the slide was then focused at 100x magnification, this brought 1cm2 area into focus the bacteria could not be seen at this magnification. To help with the identification the specimen had to be focused in at 400 x magnification the Costia is easily identified as it has a distinctive shape and the length of the flagella is used to identify this bacterium (Fig 4).
Ě…Ě…Ě… 1)2 + ∑ đ?‘Ľ2 S= √∑ đ?‘Ľ1 2 - n(đ?‘Ľ Ě…Ě…Ě… 4)2 + ∑ đ?‘Ľ4 2 - n(đ?‘Ľ
H0 There is no significant difference UV sterilization having no effect on the spread and multiplication of parasites in a system. This was the equation that was used on the data that was collected. This was used to gather the estimate standard deviation. 2
Ě…Ě…Ě… 2)2 + ∑ đ?‘Ľ3 - n(đ?‘Ľ
2
Ě…Ě…Ě… 3)2 - n(đ?‘Ľ
n+n-2 Then this equation is used to carry out the t-test out on the data that is generated from the above equation (Dytham, C. 2011.). đ?‘Ľ1 - đ?‘Ľ2 - đ?‘Ľ3 - đ?‘Ľ4 1
1
S x √đ?‘› +đ?‘›
Results After 3 weeks of monitoring and repeating the experiment measurements of the amount of parasites those are present on the specimens. Along with this the fish were monitored to check if any of the symptoms that are exhibited when the hosts become heavily infested with the bacteria. If these were to occur the experiment would have been terminated as it not ethical to put the fish through this physiological stress. The results are shown in Table 1 with averages of the parasites found on the specimens.
Figure 4 this is a photo of the identification of the parasite and counting if there is any apparent on the slide
Statistical analysis All the data that was collected when carrying out the observations was put into the unpaired t-test to prove or disprove the following:-
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Table 1 Averages of parasites found in 1cm2 sample from each host
0hrs 26.667
24hrs 11.667
48hrs 10
72hrs 4.667
Average Non UV Infected Average UV Non-infected
28.667
27.333
26.333
25
0.333
0
0
0.333
Average Non UV Non- infected
0
6
9.333
12
Average UV Infected
This table shows the average amount of parasites on the two infected fish subjected to UV sterilization and the two fish that were left untreated. Also it shows the Non-infected fish that were and were not subjected to UV sterilization. Figure 5 shows in graphical formation the correlation between UV, Non UV, infected and non infected fish.
Graph 1 Averages of Parasites counted over 3 week period 35
Infected fish (UV on) Infected fish (UV off) Non infected fish (UV on) Non infected fish (UV off)
Parasites in 1cm2
30 25 20 15 10 5 0 0
24
48
Time Period (hours)
72
Figure 6 Shows in graphical formation the correlation between UV, Non UV, infected and non infected fish with Error Bars using Standard Error
Graph 2 Averages of Parasites counted over 3 week period with Standard Error Bars 35
Infected fish (UV on) Infected fish (UV off) Non infected fish (UV on) Non infected fish (UV off)
Parasites in 1cm2
30 25 20 15 10 5 0 0
-5
24
48
72
Time Period (hours)
After carrying out the t-test on the data that was collected the critical value was 0.028 with a degree of freedom of 6 and at 5% the test statistic is below the critical value which is 2.447. This means that the null hypothesis is accepted which means there is not a significant difference between the UV sterilization being used on an infected system or not to prevent spread of the infection (Raw data in appendix 1). also is a lot less invasive type of treatment because the fish are not disturbed in any way because the UV sterilizer is attached to the filter which keeps the flow rate of the tank running. This flow of water is passed through both the inlet and outlet chamber of which contains a UV light bulb. It is a lot more effective than a normal biological filter system as much as this removes the TAN from a system the parasite can’t be broken down by the denitrifying bacteria. Whereas the UV light will break down the cellular make up of the parasite.
Discussion The results of the experiment UV sterilization have no statistical significant impact on parasite reduction. However this is not shown in the graphs that have been produced from the averages of the data over the three weeks. The graphs show that the UV light helps with the reduction and prevention of I. necatrix from spreading to the non infected fish from the infected. This was expected because UV light inhibits the parasite from reproducing which in turn decreases its number over time. With DOM also being broken down with the UV sterilizer the parasites have nothing left to feed on as they feed on the decaying epithelial cells of the host. It was expected that the non- infected fish would not get infested with the parasites because the water borne parasites would have their cells broke down by the UV light as they would pass through both the chambers where the UV light bulbs are situated. The system that did not have a UV sterilizer had a considerable amount multiplication and spreading of the parasites amongst the specimens. This was expected as there was nothing stopping the parasite from reproducing in the system. Also the water quality was monitored so that it was kept at optimum levels so there were not any other physiological stresses on the specimens. So with carrying out this experiment the UV sterilization is a vital piece of equipment to have to ensure that possible life threatening can be reduced to a non lethal level if a live stock becomes infected with this parasite. This
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Monk.N, 2010. Fish viral disease. [online]. Available at: http://www.fishchannel.com/media/fish-health/diseaseprevention/fish-viral-disease.aspx.pdf [01.05.11] Noja, E, J. 2010. Fish Disease: Diagnosis and Treatment. Iowa: Iowa State University Press Post, G. 1987. Textbook of Fish Health. Berkshire: T.F.H Publications Robertson D, A., 1985. A review of Ichthyobodo necator (Henneguy, 1883), an important and damaging fish parasite. Recent Advances in Aquaculture, 2, 1–30. Schisler G.J., Walker P.G., Chittum L.A. & Bergersen E.P., 1999. Gill ectoparasites of juvenile rainbow trout and brown trout in the upper Colorado River. Journal of Aquatic Animal Health 11, 170–174. Sommerville C. 1984. The economic importance of protozoan parasites of fish. The Fourth European Multicolloquium of Parasitology, 4, 249–250. Weaver. D.E, 1991. Importation of Diseases with Ornamental Fish Problem and Risk Analysis. [online]. Available at: http://aquafind.com/articles/importation.php [01/05/11]
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