Evaluation of the Live-Attenuated Vaccine AquaVac-COL! on Hybrid Channel x Blue Catfish Fingerlings in Earthen Ponds by Sandi Kirkland A thesis submited to the Graduate Faculty of Auburn University in partial fulfilment of the requirements for the Degree of Master of Science Auburn, Alabama December 13, 2010 Keywords: hybrid catfish, Flavobacterium columnare, Columnaris disease, vacine, aquaculture Copyright 2010 by Sandi Le Kirkland Approved by David B. Rouse, Department Head, Alumni Profesor of Fisheries and Alied Aquacultures Jefery S. Terhune, Chair, Asociate Profesor of Fisheries and Alied Aquacultures Wiliam H. Daniels, Asociate Profesor of Fisheries and Alied Aquacultures Yolanda J. Brady, Asociate Profesor of Fisheries and Alied Aquacultures ii Abstract The evaluation of the use of AquaVac- COL ? was conducted with hybrid catfish, which are a cross betwen female channel catfish (Ictalurus punctatus) and a male blue catfish (Ictalurus furcatus), to determine production rates in field conditions. Fry were stocked at rates of 25,000 fry/ha in 0.1 ha earthern ponds and cultured for 172 d in one of two treatments. The treatments (n=4) were 10-day post-hatch fish sham-vacinated and 10-day post-hatch fish vacinated with AquaVac- COL ? and then stocked in the ponds. Fingerling mean standing crop averaged 2,802 ? 268 kg/ha for sham- vacinated and 2,676 ? 424 kg/ha for vacinated treatments. No significant diferences occurred betwen treatments. Mean individual fish weight at harvest averaged 16.8 ? 0.10 g for sham- vacinated treatments and 21.1 ? 1.8 g for vacinated treatments. No significant diference for mean individual fish weight was found betwen treatments. Fed conversion ratio (FCR) averaged 2.57 ? 0.18 for the sham- vacinated treatment and 1.92 ? 0.38 for vacinated treatments and were not significantly diferent betwen treatments. Mean percent survival for the sham- vacinated treatment averaged 67.2% ? 7.2 while the vacine treatment averaged 54.2% ? 12. Observed mortality for the sham- vacinated treatments averaged 4.14% ? 1.2 while the vacine treatment averaged 4.3% ? 4.0. The study determined no significant diferences when considering the eficacy of AquaVac- COL ? in respect to production parameters betwen ponds or betwen treatments. iii Acknowledgments I want to expres my gratitude to the Auburn Universities Department of Fisheries and Allied Aquacultures. I would also like to thank specificaly Dr. Jefery Terhune for his consistent help, positive atitude, and helpful guidance during my research at Auburn. I would like to thank Dr. Bil Daniels for always being a helping hand and for teaching with pasion. Special thanks to Mark Peterman and the crew of E.W. Shel Fisheries Center for help in conducting research at the station. Love goes to my other half, Keith, who encouraged and supported me while doing my research at Auburn. iv Table of Contents Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Materials and Method. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 v List of Tables Table 1: Overal mean total amonia- nitrogen, nitrite- nitrogen, chloride, and total alkalinity (mg/L) from 0.1 ha earthen ponds with either sham vacinated or vacinated fish after 172 d. Values are means (? standard deviation; n= 4). Significance diference occurred betwen treatments if p < 0.05. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Table 2: Mean (? SD) standing crop, average weight, and FCR for hybrid catfish fry after 172 d in earthen ponds that were either vacinated or sham vacinated. Values are means (? standard deviation; n= 4). Significance diference occurred betwen treatments if p < 0.05. . . . . . . . . . . 27 Table 3: Percent survival betwen replicates ponds. No statistical comparisons were performed since replicate ponds are not paired with one another and are for presentation only. . . . . . . . . . 28 Table 4: Mean percent ? SD, observed mortality, and total percent survival for hybrid catfish during the 172 day study period from ponds stocked with either sham vacinated or vacinated fry. Values are means (? standard deviation; n= 4). Significance diference occurred betwen treatments if p < 0.05 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Table 5: Study treatments and replicate ponds within study treatments that were treated with 20- ppm formalin for Ichthyophthirius multifilis and date of first to last treatments. . . . . . . . . . . . . 30 vi List of Figures Figure 1: Wekly mean afternoon pH of ponds used to grow hybrid catfish from June 11, 2009 to November 5, 2009. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Figure 2: Daily mean temperature of ponds used to grow hybrid catfish from June 11, 2009 to November 7, 2009. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Figure 3: Average daily morning disolved oxygen levels for the vacinated and sham- vacinated treatments from 11 July ? 5 Nov 2009. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 1 INTRODUCTION Catfish production is the largest aquaculture industry in the United States. In 2009 procesed farmed- raised catfish amounted to 166 milion pounds (NAS 2010). The channel catfish, Ictalurus punctatus, is the primary species that the aquaculture industry relies upon in the United States (Goldburg et al. 2001). The most common method for the culture of channel catfish is earthen ponds at high density (Mingkang 2005). Disease is one of the most reported reasons as to loss of standing crop during the culture period. Two primary diseases afect channel catfish, enteric septicemia of catfish (ESC) and columnaris (USDA 2007a). Columnaris is a bacterium that is found in waters al over the world and is atributed to causing loss in milions of dollars per year. As the channel catfish industry is a large economic entity, reducing the disease outbreak of columnaris is of primary concern. Management practices have been used to reduce stres as the bacterium infects the organism in this state. Other methods for reducing columnaris outbreaks include treatments such as salt, potasium permanganate, copper sulfate, and hydrogen peroxide (Wakabayashi 1991). Hybrid catfish are a cross betwen female channel catfish and the male blue catfish, Ictalurus furcatus. Hybrids are commonly used in studies, as they tend to have consistently beter growth, filet yield, and harvestability (Giudice 1966; Dunham and Smitherman 1981; Smitherman et al. 1983; Dunham et al. 1987b; Dunham et al. 1990; Dunham and Brummet 1999, Bosworth et. al 2004, Tave et al. 1982; USDA 2007b; Dunham et al. 1982; Yant et. al 1976, Dunham and Argue 1998). Hybrid catfish have been observed to be les susceptible to 2 diseases, such as ESC and proliferative gil disease (Wolters et al. 1996). Although it is been observed that hybrid catfish are susceptible to columnaris, specific studies addresing susceptibility and control measures for columnaris disease in hybrid catfish have not been conducted. There are unknown diferences in disease resistance to columnaris when comparing hybrids and channel catfish. Vacinations have proven to be efective when treating various diseases. The exploration of a vacine for columnaris is stil an on- going study. A live-atenuated vacine caled AquaVac- COL ? produced by Schering Plough-Intervet, Inc. (Milsboro, Delaware) has been developed to reduce the disease effects of columnaris. Shoemaker et al. (2009) have shown AquaVac-COL ? to be safe for eyed channel catfish fry and to aid in defense against columnaris in laboratory trials only. !"#$%&"'&$()&*+,,-.&/$0"&$(12&1""3&'/$'&bas&(Micropterus salmoides floridanus)&14"56"1&/$0"&(27"8&861%&29&:"$'/&7/"3&;163<&=>;$?$5@&ABC ? &63&$&0$'&1D1'"E)& The primary question to be answered from this study was: are the production characteristics (mean survival, fed conversion ratio, individual fish weight, and final standing crop) of hybrid catfish fingerlings grown in earthen ponds from the fry to fingerling improved when using AquaVac-COL ? ? 3 LITERATURE REVIEW Channel catfish, Ictalurus punctatus, is a commonly cultured species in the United States. The development of the catfish industry has grown since the 1980s and is a fundamental contribution to the aquaculture industry in the United States. In 2004, channel catfish production grossed $480 milion. The original purpose of culturing channel catfish was for stock enhancement of streams, lakes and ponds. The development of the catfish industry occurred when advances in technology occurred and a beter understanding of the physiology of the catfish was acomplished. Spawning catfish was first achieved in 1914, which alowed catfish to be grown for stocking, and in the 1920s this was a common practice. People began to realize that catfish could be farmed for food fish, and, by the 1960s, Arkansas was producing 4000 ha of farmed catfish. The next development in the catfish industry was the fed mil in the 1970s. These developments are examples of what alowed the industry to grow, which in turn increased production significantly. By 2003, 64% of North American aquaculture production was atributed to the channel catfish, generating $348 bilion dollars (Olin 2006). The industry has also sen growth because of an increase in demand, as capture fisheries are not able to met this need. Channel catfish farming has grown with the global trend, moving from 1,000 ha in the early 1960s to 77,000 ha in 1998 (Boyd 2000). Alabama, Arkansas, and Misisippi are the main producers of catfish providing 241 milion foodsize fish on 4,856 ha in 2009. While the majority 4 of catfish provided were foodsize fish, the states also provided 877 milion fry and fingerlings grown on 5,655 ha (USDA 2009). The catfish industry has grown to the largest aquaculture industry in the United States. The amount of farm-raised catfish procesed in 2009 totaled 211 milion kg (NAS 2010). Including fed mils, procesors and supply companies, the total worth of the catfish industry is around seven bilion dollars (USDA 2007b). In 2008, the most popular farm-raised fish or seafood consumed was catfish at number six and of the 7 kg of seafood consumed by Americans, 0.4 kg were catfish (NMFS 2008). Currently, 70% of the United States aquaculture industry relies on the channel catfish (Goldburg et al. 2001). The main form of catfish culture is earthen ponds and, in some cases, high-density systems, such as cages, pens, tanks, vats, and raceways (Mingkang 2005). In-pond culture has been practiced for around 50 years and is the most succesful method for the aquaculture busines in the United States (Engle 2003). Many improvements in methods have been developed in the catfish industry. Research has been focused in areas to aid in production and technology. Improvements in hatch survival rates and protection from disease are examples of improvements in production strategies (Smal 2009; Carrias et al 2008). Nutritional studies have revealed beter methods to obtain a more profitable fed conversion ratio from fed, temperature efect, diet compositions and many other related studies (Li 2008). Improvements of in-pond grading systems have alowed for les stres on the animal and more acurate sorting method (Greenland and Gil 1972; Trimpey et. al 2004). The hybridization of a female channel catfish and a male blue catfish, I. furcatus, has proven to have more succesful culture traits when compared to channel catfish production. In 2008, over 30 milion hybrid fingerlings were produced (Li et al 2010). Hybrid catfish are bred for their superior genetics and vigor as compared to other commercialy raised catfish, the hybrid 5 dominates in culture traits (Yant et al. 1976; Dunham and Smitherman 1987a; Argue 1996; Wolters et al. 1996; Dunham and Argue 1998; Dunham and Devlin 1998). The hybrid has faster growth to market size (Giudice 1966; Dunham and Smitherman 1981; Smitherman et al. 1983; Dunham et al. 1987b; Dunham et al. 1990; Dunham and Brummet 1999). It has been shown that hybrid growth rates during food fish grow-out are 12-31% faster than channel catfish (Yant et a. 1976, Li et al 2004; Dunham et al. 2008). Market size hybrids have also been sen to reach 680- 794 g or larger when stocked at 12,355- 14,820 fish/ha (Li 2010). Compared to channel catfish, the hybrid catfish has an increased tolerance to lower disolved oxygen concentrations. A forced oxygen depletion by adding formalin to earthen ponds, cages, and concrete tanks with both channel catfish and hybrids resulted in hybrids having a 30-60% beter survival rate (Dunham et. al 1983). The hybrid has a higher catchability by seine than the channel catfish, alowing for beter harvestability (Tave et al. 1982; Dunham et al. 1982; Smitherman et al. 1983; Dunham et al. 1986, Yant et. al 1976, Dunham and Argue 1998). Higher dres out percentages have been observed in hybrids when compared to channel catfish (Argue et al. 2003). A strain of NWAC 103 line channel catfish (I. punctatus), Norris line channel catfish, and channel catfish female x blue catfish male (I. furcatus) were used to compare meat yield and meat quality traits and it was sen that the hybrid had higher dres-out and filet yields (Bosworth et. al 2004). A greater fed eficiency is sen with the hybrid. When compared to channel catfish, the hybrid has been sen to consume more fed, gain more weight, convert fed more eficiently, and have a higher net production (Li et al. 2004). Higher yield has been sen in hybrids along with an increase in body weight of 18-100% when compared to channel catfish (Smitherman et al. 1983; Dunham et al. 1987b; Dunham et al. 1990; Dunham and Brummet 1999). 6 Hybrid fry and fingerlings require more capital than other food fish, but hybrid catfish fingerlings and food fish production generaly is more profitable when compared to channel catfish at moderate prices (Li et al 2010). The most efective spawning proces for producing hybrid catfish is artificial spawning, which involves stripping female channel catfish of eggs and sacrificing a male blue catfish. Producing fry becomes expensive, as the spawning proces is more involved than channel catfish spawning. It is estimated that the increase in cost to produce a hybrid catfish fry is ! cent per inch. Therefore, where a channel catfish costs around 1! cent per inch, a hybrid catfish costs 2 cents per inch (Maser et al 1998). Due to the hybrids improved traits over the channel catfish, the additional cost should be recovered. Survivability of hybrids is greater than channel catfish as the hybrids have greater resistance to some diseases (Dunham et al. 1990, Dunham et al 2008). Hybrids have been shown to have strong resistance to primary diseases of catfish such as, enteric septicemia of catfish (ESC), caused by Edwardsiela ictaluri, motile aeromonas septicemia (Aeromonas hydrophila), Ichthyophthirius multifilis, and channel catfish virus (CCV) (Wolters et al. 1996, Maser et al 1998). Columnaris disease is a wide spread bacterial disease in culture systems. Flavobacterium columnare is the pathogen responsible for this disease and is a gram-negative rod bacterium. This disease was first described by Davis (1922), who described it as Bacilus columnaris due to the cels appearing as column-like mases, which were isolated on tisues placed in wet mounts. The organism grows as a yelow-pigmented, rhizoid bacterium when grown on Shieh agar (Decostere 1998a). The disease-causing agent was first identified from hatchery-reared sockeye salmon, Oncorhynchus nerka (Ordal and Rucker 1944). The name of the organism then changed to Chondrococcus columnaris. The name columnaris was given to the disease because of its 7 appearance when examined with a microscope. The organism appears as columns in colonies that resemble haystacks. The bacteria are 0.3 to 0.7 ?m wide x 3 to 10 ?m long (Farmer 2002). After a succesion of name changes, the current name is now Flavobacterium columnare as defined by Bernardet from molecularly identifying strain characteristics (Bernardet et al. 1996). F. columnare grows best at 25 ? C and in a temperature range from 4 to 37 o C (Amend 1982). Catfish are susceptible to columnaris at any stage of life under varying water quality situations and during al seasons at appropriate growth temperatures for the bacterium. When F. columnare infections occur, there is a 24- hour period or les for incubation and mortalities wil appear two to three days after exposure. In most cases, mortalities are dependent on temperature but can range from 10 to 100% (Holt et al 1975). While catfish are a primary species of interest from an economic stance, other fishes are susceptible to infection. Both warm water and cold water fishes are afected by columnaris. The disease is only found in fresh water and not in marine environments. The disease has been isolated from fishes in many countries around the world, including Japan, Korea, Canada, the United States, Taiwan and Europe. Many fish species can be infected, including eels (Anguila japonica and A. anguila), oriental weatherfish (Misgurnus anguilicaudatus), goldfish (Carassius auratus), common carp (Cyprinus carpio), gras carp (Ctenopharyngodon idelus), pike (Esox lucius), tench (Tincca tinca), black bullhead (Ictalurus melas Rafinesque), rainbow trout (Oncorhynchus mykis), and catfish (Wakabayashi 1991). The bacterium itself is naturaly present in warm waters around the world and is opportunistic. Therefore, if an organism becomes stresed, the bacteria are able to primarily infect the organism, which can lead to secondary infections. The commonality of this pathogen around the world and in many water environments alows it to be a key pathogen of interest in order to prevent outbreaks from occurring in culture systems. 8 Virulence factors of F. columnare are not wel known, but are known to vary with the difering genetic groups of F. columnare. The ability of F. columnare to cause an infection is due to chondroitin AC lyase that causes tisue degradation after the pathogen has atached to the organism. This enzyme deteriorates polysacharides found in the connective tisue alowing the infection to occur (Teska 1993). The efectivenes of degrading tisue varies with difering strains (Kunttu et al 2009). Virulence is also asociated with capsule size, which is produced by the bacterium. Highly virulent strains range in sizes from 120-130 nm while low virulent strains range in size from 80-90 nm (Decostere et al. 1998b). Low virulent strains have a tendency to create necrotic lesions on the gils and body alowing systemic infections to occur. Highly virulent strains tend to have more afinity towards the gils. Mortalities caused by low virulent strains are sen after a period of days while high virulent strains have mortalities occur within a 24-48 hour time period (Rucker 1953; Pacha and Ordal 1970). The common clinical signs that are asociated with columnaris are asociated with the tisue destruction that the infection causes. Signs of infection can be sen on the external portion of the fish. Usualy, lesions are a dull white or yelow color located or around the mouth, on the skin or fins, or on the gils. A typical description of columnaris is known as ?cigar mouth?, which stems from the infection causing the mouth of a diseased animal to appear as a brown color that is typical of cigars. The brown coloration wil be sen along the lips and in the internal area of the mouth cavity. Presence of columnaris can be sen when erosion of the flesh occurs around the dorsal fin and down the lateral sides, which is known as a saddleback infection (Grifin 1987). The fins can also show signs of infection through the appearance of tisue rot as the fin erodes away. The disease causes a similar appearance to external tisue, which also appears as eroded and exhibits signs of necrosis. Along with the skin and fins, the gils are a primary area 9 that the disease infects and wil also appear to be necrotic (Davis 1922). An initial diagnosis of columnaris can be determined when these signs are observed. These observations can also be confirmed for F. columnare through examination of a tisue scrap of the lesion and then examined on a wet mount under a microscope. While both low and high virulent strains are causing these clinical signs, the high virulent strain is also capable of producing high mortality without observable signs (Pacha and Ordal 1967). Once an area on an organism becomes infected, the bacteria spread quickly to tisue in the surrounding areas. There are complications with isolating F. columnare, which has inhibited its pathogenicity from being determined. In many cases, when a fish is infected with F. columnare there typicaly is a secondary infection present. The secondary infection can occur from pathogens such as Aeromonas spp., E. ictaluri, or E. tarda (Hawke and Thune 1992). Due to the secondary infection, the agent causing the primary disease is dificult to identify. Therefore, the further understanding of the pathology of F. columnare has been hindered. The isue with multiple infections is the masking of the F. columnare by a number of situations. One situation is the presence of another Flavobacterium, which could veil the presence of F. columnare as the primary cause of the infection. The presence of a combative species, such as Pseudomonas or another gram-negative bacterium, could also inhibit the finding of F. columnare (Tirola et al. 2002). The presence of these bacteria dominates F. columnare, which prevents the colonies from growing in media as a dominant species as the other species present grow more prominently. Thus, the identification of F. columnare can be overlooked and the fish misdiagnosed as to the cause of the infection. The masking of F. columnare by other species is one isue with its identification while another isue with F. columnare is the inability for it to grow on common media, such as tryptic 10 soy agar, Mueler Hinton agar, or brain heart infusion agar. Therefore, isolation and culture of F. columnare has to be conducted on a specific agar. The necesary environment for growth of this bacterium includes a low nutrient high moisture content agar. Growing the sample on Cytophaga agar was originaly conducted, but the more prominent bacteria grew more than F. columnare. The bacterium was found to resist the polymyxin B and neomycin in the agar. Adding these antibiotics to the agar alowed F. columnare to have more colonies present and prevent the growth of the combative bacterium. Shieh medium is also used to culture F. columnare and has been found to be more efective than Cytophaga agar. This type of media has a specific amount of salt that creates a more optimum growth environment specificaly for F. columnare. Shieh medium supplemented with tobramycin has been found to also be an efective method for isolating F. columnare from other bacteria found in a sample (Decostere et al. 1997). Once a diagnosis of columnaris has been confirmed, the next step is to treat the infected organism for the pathogen. There are diferent methods for treating columnaris in culture systems, but the most common are chemotheraputants. These are easily distributed in a culture system and have varying degrees of efectivenes for removing columnaris from the system. One method is to use salt as a controlling agent. An efective rate is to maintain salt levels around 3 ppt. Higher rates inhibit growth of F. columnare and clumping (Bernardet 1989). When salinity rates are at higher rates, mortalities were sen to decrease. This was atributed to the salinity preventing the pathogen from binding to the organism (Altinok and Grizle 2001). Other preventative chemotheraputants are able to reduce the presence of this particular pathogen because the form of infection is topical. Therefore, treatments of these types must be able to remove pathogens from the surface of the organism. Common methods used are chemicals such as potasium permanganate, copper sulfate and hydrogen peroxide (Wakabayashi 1991). Each 11 treatment varies depending on the size of the culture system and the rate of the active ingredient within the chemical. Treatments of a specific culture system may also have to be done multiple times in order to ensure that the pathogen has been removed from the system. Another chemical that has shown to be beneficial as a candidate for treatment of F. columnare is the herbicide Diquat, otherwise known as 6,7-dihydrodipyrido (1.2a: 2, 1-c) pyrazidinium bromide. When compared with difering levels of potasium permanganate, chloarmine-T, hydrogen peroxide and copper sulfate, Diquat was sen to efective when conducted under bath conditions (Thomas- Jinu and Goodwin 2004). Other chemicals used in aquaculture systems have also been tested to determine their efectivenes for treating columnaris. In the same study by Thomas-Jinu and Goodwin (2004), a bacterial study was conducted which resulted in al groups having no mortalities when using the drugs Terramycin ? and Romet ? . Aquaflor ? is an antibiotic that has proven efective in treating channel catfish for columnaris and has been approved by the Food and Drug Administration (FDA) (Gaunt 2010). These antibiotics can also be applied as fed. Treating infected fish with chemotheraputants, drugs or medicated fed should be avoided, if possible. The cost of treating with these methods can be an expensive option for removing a pathogen. Using certain chemicals as a management for pathogen control creates a withdrawal time in the treated organism if the organism is to be used as a food fish. The best treatment for preventing this pathogen from occurring is to manage the stres level of the culture system in order to prevent an infection from occurring. The impact disease has on cultured species causes a significant decrease in return on investment. During the culture period, many factors contribute to costs, such as a transport, labor and fed. The efect of columnaris specificaly has on cultured species can be sen worldwide. columnaris can infect many species of fish. Perhaps the most important economic repercussions 12 are sen in the catfish industry in the United States. Many farmed fish kils have been atributed to bacterial diseases. As of 2002, the diseases causing the most mortality were ESC and columnaris and 65% of fry and fingerlings deaths were atributed to these diseases by farmers. When columnaris was specificaly identified as the pathogen causing the mortalities, some producers reported losing half of the infected population (USDA 2007a). Estimations of losses due to mortalities caused by columnaris alone reach into milions of dollars per year (Schrader 2008). Culture systems, such as tanks, have been reported to have half of the population kiled when an infection from columnaris occurred (Plumb 1999). The catfish industry is economicaly important in the United States for aquaculture, but other areas of the world have negative impacts on economy due to this disease. One rainbow trout farm in Turkey reported experiencing an outbreak of columnaris and atributed it to causing 30% of the mortalities in one day (Kubilay 2008). Perch populations in England were also significantly reduced due to an outbreak of columnaris (Morley 2009). These few examples can add up to major losses economicaly when considering a worldwide combination of al mortalities caused by columnaris. This disease causes a large loss in profit if it occurs in a culture system. Therefore, it is imperative that columnaris is identified and eliminated before it is able to cause mortalities in a culture system. Vacines have played a major role in improving animal health, animal productivity and safety in public health by reducing zoonotic disease, eradicating disease in countries, and have aided in preventing spread of disease by imported animals (Frey 2007). Interest for disease prevention began in aquaculture as increases in disease were prevalent due to the ability to have higher stocking densities in culture systems. The higher densities resulted in degradation of water quality conditions and easier spread of disease. 13 Increased eforts to investigate imunology and vacination in fish began in 1935-1938 (Van Muiswinkel 2008). Protective imunity was shown against Aeromonas punctatus when injected with kiled bacteria in the laboratory (Snieszko 1938). A further study showed that upon chalenge, injection of kiled or atenuated bacteria evoked protective imunity (Schaperclaus 1942). The first published study on oral vacination was by Duff (1942), who showed that by feding cutthroat trout a diet containing chloroform-kiled bacterin (Aeromonas salmonicida) after injection or contact with clinicaly- il fish protected against furunculosis (Duff 1942). The probable first report of vacination on fish against a viral disease was done for spring viremia done by injection of formalin (Goncharov 1951). Immunization against bacterial diseases was also conducted on carp (Goncharov 1968, 1971). The prevention of vibriosis, enteric redmouth disease and furunculosis has been shown with the use of vacines (Sakai 1999). Studies have shown promising results for A. hydrophila and F. psychrophilum (Rahman 2000, Crump et al. 2005, Dumetz et al. 2007, H?gfors et al. 2008, LaFrentz et al 2008, LaPatra et al. 2010). Increased interest in live atenuated bacterial vacines began in the 1990s (Norqvist et al. 1989; Vaughan et al. 1993; Thornton et al. 1994; Lawrence et al. 1997; Hernanz Moral et al. 1998; Marsden et al. 1998; Klesius and Shoemaker 1999; Thune et al. 1999). Vacination can lower economic losses from mortalities, improve fed conversion and reduce length of time to marketability (Amend and Eshenour 1980; Tebbit et al. 1981; Horne and Robertson 1987, Lillehaug 1989, Klesius et al. 2006). Disease management for fish at a young age proves to be the most dificult in the growth stage of cultured fish (Elis 1988). During the early life stages, fish have non-specific and specific imunity. Non-specific defense mechanisms are innate factors, such as physical barriers and phagocytic cels, which help with diseases resistance. The non-specific defense system 14 prevents pathogens from atachment, invasion or replication on or in the tisue of the fish. This defense mechanism is temperature- dependent and takes more time to respond (Elis 2001). The main physical barriers are the skin and mucous. Mucous is secreted from goblet cels in the skin, gils, and gastrointestinal tract preventing colonization of microbes. Phagocytic cels function to obtain, engulf, and destroy microbes. Another form of defense is the specific defense mechanism, which develops later in life to aid long-term disease resistance. Fish naturaly have humoral imunity that consists of B cels, which are triggered when antigenic stimulation occurs causing antibodies on the surface of lymphocytes to be secreted (Katari 1992). Antibody production time is 2 to 4 weks in channel catfish, yet an infection from a pathogen could kil a fish within days. Antibody production is triggered in B cels after imunization in fish which appear in blood, bile and mucous (Wilson and Warr 1992). Live- atenuated vacines have been efectively used in livestock and poultry industries. Typicaly, atenuated vacines are used when an endemic pathogen is present in the environment of cultured fish. Therefore, atenuated vacines provide an advantage over kiled vacines in the aquatic environment. The use of modified live vacine is able to repeatedly expose the host providing protective antigens to create imunity (Shoemaker 2009). Methods for vacination of young fish are spray or bath vacination but these methods tend to be les efective than injection, which is dificult to do with young fish. Vacinating oraly has been shown to be the least efective method while vacination by injection has proven most efective (Elis 1988). Young fish can be efectively imunized via imersion with modified live vacines alowing for stimulus of the humoral imunity and long term duration of the vacine (Shoemaker 2009). Atenuated vacines, theoreticaly, can provide the capability for farmers to protect large 15 quantities of fish at a young age. Two vacines have been approved for use on 7-10 day post- hatch catfish fry to prevent ESC and columnaris. These vacines, AquaVac- ESC ? and AquaVac-COL ? , have proven to reduce mortalities for their respective diseases, and imunization from these vacines has been shown to have a duration of 1 to 2 years (Panangala et al. 2006). Vacination against columnaris has had mixed results. Pond-reared channel catfish imunized with a formalin-kiled bacterin had low-level protection from columnaris (Moore et al. 1990). Immersion imunization saw 60% protection in eels, while injection was not as efective (Mano et al. 1996). Heat and formalin- kiled preparations provided some protection for carp and eel after imersion imunization (Liewes et al. 1982, Mano et. al 1996). Oral imunization of coho salmon at 3 months old had partial protection from a heat-kiled vacine (Fujihara et al. 1971). As F. columnare causes infection by deteriorating the gils and infiltrating the body from there, a primary defense mechanism is mucus. Adhesion to gil tisue and the virulence of a strain of F. columnare are directly correlated (Decostere et al. 1999a). Adhesion on mucus- coated slides and the virulence of Finnish strains had no connection (Suomalainen et al. 2006). In another study, a les virulent strain of F. columnare mutant that forms smooth colonies had les adherence to both skin and gil tisues of channel catfish when compared to wild type strains that form hard colonies (Bader et al. 2005). Chalenged catfish were sen to have higher mortalities due to the adhesion of the F. columnare to fry (Shoemaker et al. 2007). The ability of channel catfish to fight disease at a 10-day post hatch is of question. The imune system may not fully be formed by this age. A study done with E. ictaluri to determine significant antibody generation 16 showed that humoral imune response before four weks of age in channel catfish might not be capable of making imunological tolerance (Petrie-Hanson et. al 1999). Eficacy of AquaVac-Col ? vacine in pond- reared catfish has yet to be determined. The vacine has been determined to be safe for 10-day post- hatch channel catfish fry and eficacy was sen in eyed channel catfish eggs and in10-day post hatch, 48-day post hatch, and 3 month old catfish in studies conducted in aquaria (Shoemaker et al 2009). The efectivenes of AquaVac-COL ? vacine was analyzed on 8-month old channel catfish fry 10 minutes post- vacination in which the vacine was sen to upregulate 28 expresed sequence tags that represent putative function in imune response (46%), signal transduction (21%), transcriptional regulation (11%), cel maintenance (11%), and unknown areas (11%) (Pridgeon et al. 2010). Largemouth bas fry were chalenged with AquaVac-COL ? vacine where vacinated fish were sen to have a 43% lower risk of death in one field trial utilizing large holding tanks (Bebak et al 2009). The vacine exposes the protein to an underdeveloped imune system in young fish, which may prevent the imune system from responding to the antigen if the fish is exposed to it later in life (Kunttu 2010). 17 MATERIALS AND METHODS On June 11, 2009, approximately 200,000 hybrid channel catfish fry were obtained from the Genetics unit of the E. W. Shel Fisheries Research Station, Auburn, Alabama. The hybrid strain of catfish used was Eagle strain channel females crossed with Rio Grand blue catfish. The fry were grown in the Genetics Unit and were obtained 10- d post hatch. Eight earthen ponds (0.1 ha each at a depth of 1 m) were used to grow the fry to fingerlings. The ponds were drained and dried for two weks prior to refiling from a watershed located on the research station two days before fry were stocked. Before filing, screens were placed on inlet pipes to prevent wild fish from entering the study ponds. Fry in al treatments (approximately 25,000 fry/pond) were added to the ponds on June 11, 2009. The fry were randomly separated into 2 treatments with 4 replicates per treatment. The two treatments were distinguished as a sham-vacination and vacination with AquaVac- Col ! . The fry were treated with the vacine and then placed into the earthen ponds on the same day. Briefly, fry were held in two rearing troughs until 10 days post hatch. An estimation of the number of fry stocked was based on an average weight of three samples of 25 fry each and numbers were extrapolated to estimate the total number of fry stocked. An estimated 25,000 fry were transported to each pond. Vacination of fry was carried out on the pond bank acording to the manufacture?s recommendations for use on channel catfish fry. Based on 25,000 fish, 13.0 ml of vacine was added to 4.8 L of water. The fry were submersed in the vacine solution supplied with pure oxygen provided via air stones for 2 minutes. Afterwards, an additional 4.8 L of water 18 was added to the fish holding tank for an additional 30 minutes. The fry were then placed into the ponds. In sham- vacinated control tanks, al fish underwent the same procedures minus the addition of the vacine solution. Each pond was fed twice a day by hand, once in the morning and afternoon with a commercialy- available fry fed (Purina AquaMax? Trout Diet, Gray Summit, MO). AquaMax? Fry Powder containing 50% protein and 17% lipids was used to fed for weks 1 to 4. Fish were fed with AquaMax? Fry Starter 100 containing 50% protein and 17% lipid for wek 5. AquaMax? Fry Starter 200 containing 50% protein and 17% lipid was used to fed for weks 6 to 8. AquaMax? Fingerling Starter 300 containing 50% protein and 16% lipid was used to fed for weks 9 to 13. AquaMax? Grower 400 containing 45% protein and 16% lipid was used the remaining weks of 14 to 22. Changing to a larger fed pelet was determined acording to the behavior and size development of the fry in each pond. Fish were observed after every feding and the activity of feding was recorded. Fish were fed to approximate satiation each feding based upon recommendations by Tucker and Robinson (1990), but never exceding 114 kg/ha. Disolved oxygen (DO) and temperature were recorded twice daily in the morning and afternoon at dawn and dusk, using a disolved oxygen meter (YSI model 55, YSI Inc., San Diego, California). In morning, supplemental aeration was provided to the pond. Aeration was provided nightly from evening to early morning and used emergency aeration as needed. A wekly monitoring of water quality parameters pH, temperature, chloride, total alkalinity, total amonia nitrogen, and nitrite-nitrogen was conducted from June 11, 2009 to November 30, 2009. The measurement of pH and temperature was obtained with a portable Hach Sension1 ! pH meter (Hach Chemical Company, Loveland, Colorado, USA) on the pond bank. 19 Measurement of the remaining water quality parameters were obtained with a Hach ! water quality kit (model FF-1A). Chloride levels were monitored wekly, and if observed to be below 50 ppm or 10 times the level of nitrites, salt was added to the pond. Mortalities were monitored and recorded daily. Fish found suitable for necropsy were submited to "#$%&'("#$)*"$+,%-''.$+)"/0$%1/*#%2/*$)*$%3)4'+)"'+56%7(4(+,%8,/0$+*/"56% 7(4(+,6%736%9'+%:/);,'*/*< Liver, kidney, and splen were taken from the samples and plated on brain heart infusion (BHI) agar media and Hsu-Shots agar media. To determine the cause of the disease and confirmation of the causative pathogen, biochemical testing of isolated bacterial colonies were run. Skin scrapes and gil samples were observed for parasite infestations. Confirmation of the causative disease gave direction to determining the treatment, if any, for the infected pond. Harvesting of the ponds began on November 30, 2009, and ended on December 4, 2009, in which fingerling fish were collected after 5 months of growing in the ponds. Al fish harvested were weighed in bulk on a standard industrial bench scale (0.0001 lb readability) to determine the final standing crop of each pond. Subsamples from each pond, three 100 fish samples, were used to calculate average weight of the fish and then projected out to determine final numbers of fish harvested. Observed mortalities, total percent survival, and fed conversion ratio were calculated. &")"/*"/=)>%),)>5*/*%'9%)>>%.)+)?$"$+*%@)*%=',:(="$:%(*/,;%)% .)/+$:%*)?.>$%!""$*"%)=='+:/,;%"'%A)++%BCDDDE6%@#$+$%*/;,/9/=),=$%@)*%#$>:%)"%.%? %F()"/,;%"#$%$99/=)=5%),:%4$,$9/"*%'9%(*/,;%AquaVac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quaVac-COL ? have been conducted only in laboratory setings and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quaVac-COL ? %#)*%4$$,%*$$,%"'%4$%$99$="/0$%/,%=#),,$>% =)"9/*#%9+'?%CFP:)5%.'*"%#)"=#%"'%X%?',"#%'>:%B&#'$?)N$+%$"<%)>%LFFDE<%O'@$0$+6%@/"#%"#/*% ='>(?,)+/*%0)==/,$%4$/,;%)..>/$:%"'%9/*#%)"%CFP%:%.'*"%#)"=#6%/"%/*%(,N,'@,%#'@%>',;%"#$% 0)==/,$%'+;),/*?%+$*/:$*%/,%"#$%9/*#%"'%)>>'@%$,'(;#%'9%),%$I.'*(+$%"'%*"/?(>)"$%"#$% /??(,$%*5*"$?%BW/*$%),:%R$+#(,$%LFFCE<%O54+/:%=)"9/*#%#)0$%5$"%"'%4$%:/+$=">5%*"(:/$:% 24 @/"#%AquaVac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ybrid catfish have advantages over channel catfish when comparing production parameters. The ability to have beter fed conversion ratio, growth to market size, and higher dres- out percentages (Li et al. 2004, Giudice 1966; Dunham and Smitherman 1981; Smitherman et al. 1983; Dunham et al. 1987b; Dunham et al. 1990; Dunham and Brummet 1999; Argue et al. 2003). Although hybrids have greater resistance to certain diseases, there is stil more information to be discovered in respect to determining if hybrids are more resistant to columnaris compared to channel catfish (Wolters et al. 1996), as wel as other physiological stres responses that may make the fish susceptible to disease. % 25 % 7>"#'(;#%"#$%='*"%'9%#54+/:*%?)5%4$%?'+$%$I.$,*/0$%)"%"#$%',*$"%'9%.+':(="/',6%the most efective spawning proces for producing hybrid catfish is artificial spawning, which involves stripping female channel catfish of eggs and sacrificing a male blue catfish. This action drasticaly increases labor cost for producing hybrid catfish. It is estimated that the increase in cost to produce a hybrid catfish fry is $0.025 per inch (Maser et al 1998). The cost of the AquaVac-COL ? should be considered in farmers? economic outlooks. The possibility of discounts for fry producers to buy the vacine is around $3000-5000 / milion fry adding an additional $0.005 per fish to production cost (Kasha Cox, personnel communication, Schering Plough-Intervet, Inc.). R#$%*"(:5%=),%4$%*$$,%)*%)%./>'"%*"(:5%9'+%"#$%AquaVac-COL ? 0)==/,$%',%#54+/:% =)"9/*#%/,%"#$%9/$>:<%['%*")"/*"/=)>%:/99$+$,=$*%@$+$%*$$,%/,%"#/*%$I.$+/?$,"%/,%+$>)"/',%"'% ='?.)+/,;%"#$%4$,$9/"*%'9%0)==/,)"$:%9/*#%@/"#%AquaVac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able 1: Overal mean total amonia- nitrogen, nitrite- nitrogen, chloride, and total alkalinity (mg/L) from 0.1 ha earthen ponds with either sham vacinated or vacinated fish after 172 d. Values are means (? standard deviation; n= 4). Significance diference occurred betwen treatments if p < 0.05. Treatment Total Amonia Nitrogen Nitrite Nitrogen Chloride Total Alkalinity Sham- Vacinated 1.34 ? 0.15 0.085 ? 0.02 45 ? 1.87 48 ? 1.23 Vacinated 1.32 ? 0.27 0.085 ? 0.01 48 ? 2.01 45 ? 1.63 p-value 0.97 1 0.067 0.13 27 Table 2: Mean (? SD) standing crop, average weight, and FCR for hybrid catfish fry after 172 d in earthen ponds that were either vacinated or sham vacinated. Values are means (? standard deviation; n= 4). Significance diference occurred betwen treatments if p < 0.05. Treatment Standing Crop (kg/ha) Ave. Weight (g) FCR Sham- Vacinated 2802 ? 268 16.8 ? 0.99 2.57 ? 0.18 Vacinated 2676 ? 424 21.1 ? 1.8 2.92 ? 0.38 P-value 0.77 0.99 0.37 28 Table 3: Percent survival betwen replicates ponds. No statistical comparisons were performed since replicate ponds are not paired with one another and are for presentation only. Replicate Treatment 1 2 3 4 Mean Sham- Vacinated 56% 55% 85% 73% 67 ? .07 Vacinated 52% 44% 92% 29% (37.3%) 54 ? .12 (56.3) 29 Table 4: Mean percent ? SD, observed mortality, and total percent survival for hybrid catfish during the 172 day study period from ponds stocked with either sham vacinated or vacinated fry. Values are means (? standard deviation; n= 4). Significance diference occurred betwen treatments if p < 0.05. Treatment Observed Mortality (%) Survival (%) Sham- Vacinated 4.14 ?1.2 67.2 ? 7.2 Vacinated 4.3 ? 4 54.2 ? 13 P-value 0.96 0.32 30 Table 5: Study treatments and replicate ponds within study treatments that were treated with 20- ppm formalin for ()*!*+&,*!*#-#./%0.1!#2#1##/%and date of first to last treatments. Treatment Date Beginning Treated Date End Treated Number of Treatments Sham Vacinated Replicate 3 24 Sept 2009 1 Oct 2009 3 Vacinated Replicate 1 24 Sept 2009 29 Sept 2009 2 Replicate 4 29 Sept 2009 Replicate 2 30 Oct 2009 3 Nov 2009 3 31 Figure 1: Wekly mean afternoon pH of ponds used to grow hybrid catfish from June 11, 2009 to November 5, 2009. 32 Figure 2: Daily mean temperature of ponds used to grow hybrid catfish from June 11, 2009 to November 7, 2009 33 Figure 3: Average daily morning disolved oxygen levels for the vacinated and sham- vacinated treatments from 11 July ? 5 Nov 2009. 34 LITERATURE CITED Altinok, I., and J. M. Grizle. 2001. Efects for brackish water on growth, fed conversion and energy absorption eficiency by juvenile euryhaline and freshwater stenohaline fishes. Journal of Fish Biology 59: 1142-1152. Amend, D.F. and R.W. Eshenour. 1980. Development and use of commercial fish vacines. Salmonid (March/April): 8-12. Amend, D. F. 1982. 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