EVALUATION?OF?AN?AQUAVAC?ESC???BOOSTER?ON?PRODUCTION?OF?FOOD?SIZE?CHANNEL?CATFISH?Ictalurus?punctatus?
IN?EARTHERN?PONDS?
Except?where?reference?is?made?to?the?work?of?others,?the?work?described?in?this?thesis?is?my?own?or?was?done?in?colaboration?with?my?advisory?comitee.?This?thesis?does?not?include?proprietary?or?classified?information. 
???????????????????Joshua?P.?McNeely?
Certificate?of?Approval: 
?????????????? ???????????????David?R.?Rouse??????????????????????????????????????????????????????????????Jeffery?S.?Terhune,?Chair?Profesor? Asistant?Professor?Fisheries?and?Allied?Aquacultures? Fisheries?and?Allied?Aquacultures 
?????????????? ???????????????William?H.?Daniels????????????????????????????Jesse?A.?Chappell?Associate?Profesor? Asistant?Professor?Fisheries?and?Allied?Aquacultures? Fisheries?and?Allied?Aquacultures?
_____________________________?Joe?F.?Pitman?Interim?Dean?Graduate?School
EVALUATION?OF?AN?AQUAVAC?ESC???BOOSTER?ON?PRODUCTION?OF?FOOD?SIZE?CHANNEL?CATFISH?Ictalurus?punctatus?
IN?EARTHERN?PONDS?
Joshua?P.?McNeely?
A?Thesis?Submited?to?
the?Graduate?School?of?Auburn?University?
in?Partial?Fulfillment?of?the?Requirements?for?the?
Degree?of?Master?of?Science?
Auburn,?Alabama?December?15,?2006
iii?
EVALUATION?OF?AN?AQUAVAC?ESC???BOOSTER?ON?PRODUCTION?OF?FOOD?SIZE?CHANNEL?CATFISH?Ictalurus?punctatus?
IN?EARTHERN?PONDS?Joshua?P.?McNeely?
Permision?is?herewith?granted?to?Auburn?University?to?make?copies?of?this?thesis?at?its?discretion,?upon?the?request?of?individuals?or?institutions?and?at?their?expense.?The?author?reserves?al?publication?rights. 
????????????????Signature?of?Author 
????????????????Date?of?Graduation
iv?
VITA?
Joshua?Paul?McNeely,?son?of?Paul?and?Kathy?McNeely,?was?born?in?Houston,?Texas?on?June?4,?1980.?He?attended?Splendora?High?Schol?in?Splendora,?
Texas,?and?graduated?in?1998.?In?August?2002,?he?earned?a?Bachelor?of?Science?degree?in?Wildlife?and?Fisheries?Sciences?from?Texas?A&M?University.?He?married?Elizabeth?
Grace?Adams,?daughter?of?Michael?and?Ingrid?Adams,?on?July?12,?2003.?After?teaching?6?th?grade?science?at?Splendora?Middle?Schol?for?two?years,?he?entered?graduate?schol?in?
August?2004?to?pursue?a?Master?of?Science?degree?in?Aquaculture?at?Auburn?University.
v?
THESIS?ABSTRACT?EVALUATION?OF?AN?AQUAVAC?ESC???BOOSTER?ON?PRODUCTION?OF?
FOOD?SIZE?CHANNEL?CATFISH?Ictalurus?punctatus?IN?EARTHERN?PONDS?
Joshua?P.?McNeely?Master?of?Science,?December?15,?2006?(B.S.,?Texas?A&M?University,?2002)?
95?Typed?Pages?Directed?by?Jefery?S.?Terhune?
The?efect?of?administering?a?more?economically?feasible?dose?of?AQUAVAC??ESC???to?channel?catfish?(Ictalurus?punctatus)?fingerlings?for?protection?against?enteric?
septicemia?of?catfish?(ESC)?was?evaluated.??The?study?consisted?of?three?laboratory?components?and?a?field?study.?
In?the?first?laboratory?study,?fish?were?vaccinated?by?immersion?at?24?C?at?2.44?x?10?4?,?3.48?x?10?5?,?or?3.16?x?10?6?CFU/mL?for?4?or?8?hrs.?Serum?samples?were?later?
colected?and?evaluated?for?agglutinating?antibody?titers?against?Edwardsiella?ictaluri,?the?causative?agent?of?ESC.?Mean?antibody?titers?were?significantly?higher?in?fish?vaccinated?
at?3.48?x?10?5?CFU/mL?than?in?fish?vaccinated?at?2.44?x?10?4?CFU/mL.?In?addition,?antibody?titers?were?significantly?higher?in?fish?vaccinated?at?3.16?x?10?6?CFU/mL?than?in
vi?
fish?vaccinated?at?the?lower?doses.?The?main?efect?of?time,?however,?was?not?found?to?be?significant,?nor?was?a?dose*time?interaction?observed.?
In?the?second?laboratory?study,?fingerlings?were?stocked?into?aquaria,?vaccinated?by?immersion?at?18?C?at?1.0?x?10?4?,?1.0?x?10?5?,?or?1.0?x?10?6?CFU/mL?for?4?h,?and?later?
challenged?withE.?ictaluri.?No?significant?diferences?in?antibody?titers?were?observed?among?treatments.?
In?the?third?laboratory?study,?fingerlings?were?stocked?into?aquaria,?vaccinated?by?immersion?at?25?C?at?0,?1.95?x?10?4?,?3.80?x?10?5?,?or?1.95?x?10?6?CFU/mL?for?4?h,?and?later?
challenged?withE.?ictaluri.?Significant?diferences?were?observed?between?control?fish?and?fish?vaccinated?at?the?highest?dose?and?between?fish?vaccinated?at?the?highest?dose?
and?fish?vaccinated?at?the?lowest?dose.?In?the?field?study,?treatment?groups?consisted?of?fish?(1)?non?vaccinated?as?fry?and?
non?vaccinated?as?fingerlings,?(2)?vaccinated?as?fry?at?10?d?post?hatch?and?non?vaccinated?as?fingerlings,?(3)?non?vaccinated?as?fry?and?vaccinated?as?fingerlings,?and?(4)?vaccinated?
as?fry?at?10?d?post?hatch?and?vaccinated?as?fingerlings.?Fish?were?vaccinated?by?immersion?at?18?C?for?4?h?at?a?concentration?1:10?dilution?rate?of?the?manufacturer?s?
recomended?dose?rate.?The?main?efect?of?vaccination?at?the?fry?stage?on?survival?was?found?to?be?significant.?However,?the?main?efect?of?vaccination?at?the?fingerling?stage?
on?survival?was?not?found?to?be?significant,?nor?was?a?fry*fingerling?interaction?observed.?The?main?efects?of?vaccination?at?the?fry?stage?and?vaccination?at?the?fingerling?stage?on?
FCR?values,?average?weights,?or?net?production?figures?were?not?found?to?be?significant,?nor?were?any?fry*fingerling?interactions?observed.
vii?
ACKNOWLEDGMENTS?
The?author?would?like?to?thank?Dr.?Jeffery?Terhune?for?his?invitation?to?conduct?research?in?the?area?of?fish?health,?his?patience?in?explaining?certain?aspects?of?the?project?
time?and?again,?and?his?flexibility?in?allowing?his?students?to?pursue?their?specific?career?objectives.?He?would?also?like?to?thank?Chris?Sayles?for?alleviating?some?of?the?burdens?
associated?with?the?project?and?Dr.?Edzard?Van?Santen?for?assisting?with?statistical?aspects?of?the?project.?A?special?thanks?goes?to?his?parents,?Paul?and?Kathy?McNeely,?
whose?financial?support?allowed?him?to?pursue?a?master?s?degree.??The?author?would?like?to?dedicate?this?work?to?Elizabeth?McNeely,?his?wife?and?best?friend,?whose?love?and?
support?spurred?him?on?throughout?his?two?and?a?half?years?of?study.
viii?
Style?manual?or?journal?used:?Journal?of?the?World?Aquaculture?Society?Computer?software?used:?Microsoft?Word?2000,?Microsoft?Excel?2000,?SAS?9.1.
ix?
TABLE?OF?CONTENTS?LIST?OF?TABLES?.? .? .? .? .? .? .? .? .????xi?
LIST?OF?FIGURES?.? .? .? .? .? .? .? .? .???xii?I.? INTRODUCTION?.? .? .? .? .? .? .? .?????1?
I.? LITERATURE?REVIEW?.? .? .? .? .? .? .?????4?A.?The?Disease?.? .? .? .? .? .? .? .?????4?
B.?The?Pathogen?.? .? .? .? .? .? .? .?????5?C.?Epidemiology?.? .? .? .? .? .? .? .?????6?
D.?Imune?System?of?Channel?Catfish?.? .? .? .? .????11?E.?Factors?Afecting?the?Imune?System?.? .? .? .? .????18?
F.?Imune?Response?of?Channel?Catfish?to?E.?ictaluri? .? .? .????23?G.?Control?Methods?.? .? .? .? .? .? .? .????29?
H.?Development?of?a?Vaccine.?.? .? .? .? .? .????32?I.?Oral?and?Imersion?Bacterin?Vaccine?Trials.? .? .? .? .????33?
J.?Virulent,?Live?Vaccine?Trials? .? .? .? .? .? .????36?K.?Attenuated,?Live?Vaccine?Trials?.? .? .? .? .? .????37?
II.? MATERIALS?AND?METHODS?.? .? .? .? .? .????44?A.?Laboratory?Experiment?1?.? .? .? .? .? .? .????44?
B.?Laboratory?Experiment?2?.? .? .? .? .? .? .????45
x?
C.?Laboratory?Experiment?3?.? .? .? .? .? .? .????46?D.?Pond?Study?.? .? .? .? .? .? .? .????47?
E.?Statistical?Analyses.? .? .? .? .? .? .? .????50?IV.? RESULTS?.? .? .? .? .? .? .? .? .????52?
A.?Laboratory?Experiment?1?.? .? .? .? .? .? .????52?B.?Laboratory?Experiment?2?.? .? .? .? .? .? .????52?
C.?Laboratory?Experiment?3?.? .? .? .? .? .? .????53?D.?Pond?Study?.? .? .? .? .? .? .? .????53?
V.? DISCUSSION?.? .? .? .? .? .? .? .????63?VI.? LITERATURE?CITED?.? .? .? .? .? .? .????73
xi?
LIST?OF?TABLES?
1.? Mean?antibody?titers?to?E.?ictaluriof?variable?vaccine?dose?and?immersion?time?trial?conducted?at?24?C????.??????.??????.??????.??????.?55?
2.? Mean?survival?rates?of?fingerlings?vaccinated?with?AQUAVAC?ESC?for?4?h?at?variable?doses?.??????.?????.??????.??????.?????.??????.??????.? 57?
3.? Mean?survival?rates,?FCR,?average?final?weights,?and?net?production?of?vaccinated?fod?fish?trial????.??????.??????.??????.??????.?????.??????.? 58?
4.? Mean?values?of?TAN,?pH,?nitrite,?and?chloride?during?vaccinated?fod??fish?trial????.??????.? .??????.?????.??????.??????.?????.??????.? 59?
5.? Mean?values?of?morning?and?evening?DO,?total?hardnes,?and?total?alkalinity?during?vaccinated?fod?fish?trial??.??????.??????.? .?????.? 60?
6.? Mean?number?of?times?disolved?oxygen?fell?below?2.5?ppm?and?pH?rose?above?9.5?in?catfish?ponds?stocked?at?20,000?fish/ha.??????.??????.?????.? 61
xii?
LIST?OF?FIGURES?
1.? Mean?antibody?titers?to?E.?ictaluriof?fingerlings?vaccinated?with?AQUAVAC?ESC?at?three?diferent?doses?for?4?h?at?18?C??.??????.?????.? 56?
2.? Overall?change?of?average?morning?and?afternon?water?temperature?(?C)?in?the?catfish?ponds?stocked?at?20,000?fish/ha??????.??????.??????.?????.? 62
1?
INTRODUCTION?
Enteric?Septicemia?of?Catfish?(ESC)?is?the?most?prevalent?disease?afecting?the?comercial?catfish?industry?(USDA?2003a,b).?In?past?years,?ESC?has?been?reported?to?
cause?annual?direct?loses?to?the?industry?totaling?$20?30?million?(Plumb?and?Vinitnantharat?1993).?In?1981,?Hawke?et?al.?characterized?and?named?the?causative?agent?
of?ESC?Edwardsiella?ictaluri.?The?disease?primarily?afects?young?fingerlings?in?the?fall.?However,?fingerlings?that?have?not?survived?severe?outbreaks?in?their?first?fal?may?be?
highly?susceptible?once?they?are?stocked?into?fod?fish?ponds.?Fish?farmers?have?attempted?to?control?ESC?outbreaks?by?ofering?medicated?feed?
or?by?restricting?the?amount?of?feed?ofered?(Wise?and?Johnson?1998).?Romet???(a?5:1?mixture?of?sulfadimethoxine?and?ormetoprim)?and?Aquaflor???(florfenicol)?are?the?only?
antibiotics?approved?by?the?Fod?and?Drug?Administration?(FDA)?for?treating?ESC?(Gaunt?et?al.?2003).?However,?antibiotic?treatment?is?expensive?and?fish?afected?with?
ESC?typically?reduce?their?feeding?activity,?leading?to?antibiotic?delivery?problems?(Klesius?and?Shoemaker?1998).?In?addition,?plasmid?mediated?resistance?of?several?
strains?ofE.?ictalurito?Romet?has?been?reported?(Starliper?et?al.1993).?Restricting?feed?may?control?ESC,?but?may?lead?to?a?los?of?production.?
Due?to?lack?of?eficient?control?measures,?interest?in?developing?a?vaccine?for?ESC?begangrowing?in?the?late?1980s.?Bacterins?were?developed?(Shoemaker?and?Klesius
2?
1997),?but?they?failed?to?consistently?provide?protection?(Thune?et?al.?1994??Plumb?et?al.?1994??Shoemaker?and?Klesius?1997).?Lack?of?exposure?to?or?uptake?of?bacterins?may?
have?contributed?to?their?inconsistent?success?(Ellis?1988??Thune?et?al.?1994??Shoemaker?and?Klesius?1997??Nusbaum?and?Morison?1996).?However,?the?most?significant?
contributing?factor?was?probably?their?limitation?in?producing?only?a?humoral?immune?response,?which?may?not?necessarily?provide?protection?(Klesius?and?Sealy?1995??Plumb?
et?al.?1986,?1994??Wolters?et?al.?1996??Thune?et?al.?1997).?Due?to?the?failure?of?bacterins?to?provide?consistent?protection,?the?search?for?an?
attenuated,?live?vaccine?was?begun.?Unlike?bacterins,?live?vaccines?are?capable?of?inducing?cell?mediated?immunity?(Klesius?1992a).?In?time,?Klesius?and?Shoemaker?
(1998)?produced?a?live,?attenuatedE.?ictaluri?vaccine?(E.?ictaluri?RE?33)?that,?when?administered?by?immersion,?could?protect?juvenile?channel?catfish?3?9?mos?of?age?for?at?
least?4?mos.?Intervet?Inc.???licensed?and?produced?the?vaccine?under?the?name?AQUAVAC?ESC???(Shoemaker?et?al.?2002).?The?AQUAVAC?ESC?vaccine?has?been?
shown?to?provide?protection?when?administered?to?fry?as?young?as?7?d?(Shoemaker?et?al.?1999).?
Evaluation?of?the?AQUAVAC?ESC?vaccine?under?experimental?pond?conditions?has?shown?mixed?results.?Wise?et?al.?(2000)?vaccinated?channel?catfish?fingerlings?72?d?
post?hatch?at?1.0?x?10?7?colony?forming?units?CFU/mL,?and?later?challenged?them?in?the?laboratory?and?in?the?field?in?floating?net?pens.?In?both?the?field?and?laboratory?
challenges,?survival?was?significantly?higher?in?vaccinated?fish?than?in?control?fish.?However,?data?from?a?study?by?Carrias?(2005),?which?evaluated?under?normal?pond?
conditions?the?efect?of?vaccinating?fry?with?AQUAVAC?ESC?on?survival,?was
3?
inconclusive.??Carrias?(2005)?vaccinated?channel?catfish?fry?at?10?d?post?hatch?or?32?d?post?hatch?and?stocked?them?into?primary?nursery?tanks?for?1?mo?prior?to?being?stocked?
into?ponds.?The?fish?were?then?grown?to?the?fingerling?stage.?Survival?of?32?d?vaccinates?was?significantly?higher?than?controls,?but?survival?of?10?d?vaccinates?was?not?
significantly?higher.?Because?the?study?was?conducted?under?pond?conditions,?unknown?factors?that?also?contribute?to?fish?mortalities?may?have?obscured?a?statistically?significant?
diference.?To?date,?no?studies?have?evaluated?the?relative?survival?of?channel?catfish?
immunized?with?AQUAVAC?ESC?as?fingerlings?under?pond?conditions.?The?volume?of?vaccine?required?to?administer?it?to?fingerlings?at?the?dose?currently?recomended?by?the?
manufacturer?is?cost?prohibitive.??However,?it?may?be?posible?to?substitute?a?longer?immersion?time?(4?to?8?h?as?compared?to?the?standard?protocol?of?17?min)?for?a?lower?
dose.?Administering?the?vaccine?to?fingerlings?would?be?beneficial?in?that?it?may?alow?farmers?the?option?of?vaccinating?smal?fingerlings?while?moving?them?to?fod?fish?ponds?
or?stocker?phase?ponds.?It?may?also?alow?the?administration?of?a?boster?vaccine?to?fingerlings?previously?vaccinated?as?fry.??The?objectives?of?this?study?were?to?
(1)?evaluate?the?ability?of?lower?vaccine?doses?administered?at?longer?exposure?times?to?confer?protection?in?channel?catfish?fingerlings?against?ESC,?(2)?evaluate?under?normal?
pond?conditions?the?relative?survival?of?fish?immunized?as?fry?with?AQUAVAC?ESC?and?given?a?boster?of?the?vaccine?as?fingerlings,?and?(3)?begin?evaluating?the?long?term?
benefits?of?vaccination?to?determine?if?a?boster?is?necessary?for?life?long?protection.
4?
LITERATURE?REVIEW?
Enteric?Septicemia?of?Catfish?(ESC)?is?the?most?prevalent?disease?afecting?the?comercial?catfish?industry?and?causes?the?most?economic?los?to?the?industry.?In?2002,?
ESC?was?reported?to?have?caused?loses?on?53%?of?fry/fingerling?operations?and?61%?of?fod?size?fish?operations?(USDA?2003a,b).?In?past?years?ESC?has?been?reported?to?cause?
annual?direct?loses?to?the?industry?totaling?$20?30?million?(Plumb?and?Vinitnantharat?1993).?
The?Disease?The?first?cases?of?ESC?were?documented?at?the?Southeastern?Cooperative?Fish?
Disease?Laboratory?of?Auburn?University?in?1976?in?channel?catfish?(Ictalurus?punctatus).?Hawke?(1979)?first?described?clinical?signs?associated?with?ESC,?including:?
ascites??necrosis?of?the?liver??petechial?hemorhaging??exophthalmia??small?cutaneous?lesions??and?a?large?open?lesion?between?the?eyes,?which?gives?ESC?its?comon?name,?
?hole?in?the?head?disease?.?Behavioral?signs?of?ESC?include:?anorexia,?tail?chasing,?hanging?at?the?surface?tail?down,?and?spiraling.?
ESC?can?present?itself?in?either?an?acute?or?chronic?form.?However,?the?acute?form?is?more?comon?(Klesius?1992a??Newton?et?al.?1989).?High?mortalities?and?general?
septicemia?typically?characterize?the?acute?form,?whereas?low?mortalities?and??hole?in??the?head??typically?characterize?the?chronic?form?(Newton?et?al.?1989).?Fingerlings
5?
generally?sufer?from?the?acute?form,?whereas?fod?fish?generally?sufer?from?the?chronic?form?(Wise?et?al.?2004).?
The?Pathogen?When?the?first?cases?of?ESC?were?investigated,?a?bacterium?was?suspected?to?be?
the?causative?agent.?Koch?s?postulates?were?used?to?confirm?that?the?suspect?bacterium?was?indeed?causing?the?disease?(Hawke?1979).?Microscopy?and?biochemical?
characterization?of?the?bacterium?by?Hawke?(1979)?indicated?that?the?causative?agent?of?ESC?belonged?to?family?Enterobacteriacae.?Microscopy?revealed?that?the?bacterium?was?
Gram?negative?and?rod?shaped.?Biochemical?characterization?revealed?that?isolates?were?cytochrome?oxidase?negative,?fermentive?in?glucose?O/F?medium,?and?reduced?nitrate?to?
nitrite.?With?a?few?exceptions,?al?members?of?Enterobacteriacae?share?these?characteristics?(Holt?1994).?
Use?of?DNA:DNA?hybridization?technique?indicated?that?the?causative?agent?of?ESC?was?more?closely?related?to?Edwardsiella?tardathan?any?other?member?of?
Enterobacteriacae?(Hawke?1979).?Later?work?by?Zhang?and?Arias?(2006),?in?which?a?bootstrap?analysis?was?used?to?produce?a?phylogenetic?tree?based?on?23S?rRNA?
sequences,?confirmed?the?close?relationship.?The?organism?difered?fromE.?tarda?in?that?it?was?negative?for?hydrogen?sulfide?production?and?the?indole?biochemical?test?and?was?
nonmotile?at?37?C.?Therefore,?the?Center?for?Disease?Control?in?Atlanta,?Georgia?considered?the?organism?a?new?species?in?the?genus?Edwardsiella?(Hawke?1979).?In?
1981,?Hawke?et?al.?fully?characterized,?clasified,?and?named?the?bacteriumEdwardsiella?ictaluri.
6?
Epidemiology?In?the?Southeastern?United?States,?where?the?channel?catfish?industry?is?
concentrated,?epizootics?of?ESC?comonly?occur?during?late?spring?and?early?fall?due?to?favorable?temperatures?for?bacterial?growth?and?reproduction.?The?acute?form?of?ESC?
tends?to?occur?when?temperatures?are?between?22?C?and?28?C,?while?the?chronic?form?tends?to?occur?when?temperatures?are?between?18?C?and?22?C?or?higher?than?28?C.?Below?
18?C?or?above?30?C,?ESC?outbreaks?are?rare?(Tucker?and?Robinson?1990).?Temperature?ranges?asociated?with?both?the?acute?and?chronic?forms?corelate?wel?with?the?optimum?
growth?temperature?for?E.?ictaluri,?which?is?between?25?C?and?30?C?(Hawke?et?al.?1981),?but?these?temperature?ranges?are?mere?generalizations.?Wise?et?al.?(1997)?exposed?
channel?catfish?fingerlings?to?E.?ictaluriin?aquaria,?and,?after?holding?the?fish?at?a?constant?temperature?(26?28?C)?for?216?d,?necropsied?fish?surviving?acute?infection.?The?
researchers?were?able?to?isolate?E.?ictaluri?from?the?posterior?kidney?or?brain?of?a?small?percentage?of?fish?having?cleared?the?infection?from?the?blod?and?may?have?been?able?to?
isolate?the?bacterium?from?a?larger?percentage?of?fish?had?they?used?enrichment?techniques.?The?study?demonstrated?that?chronic?infections?can?develop?in?fish?surviving?
acute?infections,?even?if?the?temperature?is?held?constant?within?the?range?typically?associated?with?acute?infections.?
E.?ictaluri?primarily?infects?channel?catfish?and?especially?causes?severe?economic?loses?in?na?ve,?young?of?the?year?fry?and?fingerlings?in?the?fall?of?the?year.?However,?the?
bacteriumhas?been?shown?to?naturally?infect?walking?catfish?Clarias?batrachus,?blue?catfishIctalurus?furcatus,?white?catfishAmeiurus?catus,?and?brown?bullhead?Ameiurus?
nebulosus?as?well?(Tucker?and?Robinson?1990).?An?aquatic?assessment?of?the?Cohansey
7?
River?in?New?Jersey?revealed?that?tadpole?madtoms?Noturus?gyrinus?are?also?susceptible?to?natural?infection?(Klesius?et?al.?2003).?Although?no?reports?have?ben?made?ofE.?
ictaluri?causing?natural?infections?in?blue?tilapia?Oreochromis?aureus,?chinok?salmon?Oncorhynchus?tshawytscha,?or?rainbow?trout?Oncorhynchus?mykis,?these?species?have?
been?experimentally?infected?with?the?bacterium?(Plumb?and?Sanchez?1983??Baxa?and?Hedrick?1989).?
E.?ictaluri?has?been?shown?to?survive?in?pond?water?for?short?periods?of?time?and?pond?mud?for?longer?periods?of?time.??Plumb?and?Quinlan?(1986)?inoculated?sterile?pond?
water?samples?withE.?ictaluri?and?incubated?them?at?25?C.?Samples?of?pond?mud?were?prepared?in?the?same?way,?but?were?incubated?at?18?C?as?wel.?After?incubation,?samples?
were?assayed?for?the?presence?ofE.?ictalurion?designated?days.?The?bacterium?survived?for?only?15?d?in?the?water?sample,?but?survived?40?d?in?mud?incubated?at?18?C?and?95?d?in?
mud?incubated?at?25?C.?The?researchers?concluded?that?E.?ictaluri?is?probably?not?a?strict?obligate?pathogen,?but?that?only?certain?environments?will?support?its?survival?outside?
channel?catfish.?Channel?catfish?have?ben?shown?to?serve?as?cariers?ofE.?ictaluri?after?surviving?
infection?byE.?ictaluri,?even?if?treated?with?antibiotics.?Mqolomba?and?Plumb?(1992)?experimentally?infected?channel?catfish?and?later?attempted?to?recover?the?bacterium?from?
surviving?fish.?E.?ictaluri?was?present?in?tisues?of?asymptomatic?channel?catfish?81?d?post?infection.?Klesius?(1992b)?ofered?fingerlings?known?to?have?sufered?from?clinical?
ESC?feed?containing?Romet?30?or?non?medicated?feed.?Periodically?over?a?270?d?period,?the?trunk?kidney?was?tested?for?the?presence?ofE.?ictaluri?and?antibody?titers.?In?fish?
given?medicated?feed,?the?bacterium?was?detected?at?90?d?and?270?d?and?serum?antibodies
8?
to?E.?ictaluri?were?detected?at?30?d?and?270?d.?A?greater?percentage?of?fish?contained?E.?ictaluri?at?270?d?than?at?90?d,?and?a?greater?percentage?of?fish?tested?positive?for?
antibodies?at?270?d?than?at?30?d.?Klesius?(1992b)?hypothesized?Romet?30?failed?to?killE.?ictaluri?within?macrophages,?leading?to?establishment?of?a?carrier?state.?
E.?ictaluri?can?be?transmited?in?a?variety?of?ways.?Shotts?et?al.?(1986)?placed?channel?catfish?fingerlings?exposed?to?E.?ictaluri?in?tanks?with?unexposed?fish.?Some?of?
the?fish?unexposed?to?E.?ictaluri?by?the?researchers?developed?systemic?infections,?thus?demonstrating?that?horizontal?transmision?can?occur.?Horizontal?transmision?may?also?
occur?when?fish?eat?infected?carcasses?(Tucker?and?Robinson?1990)?or?by?the?shedding?of?viable?E.?ictaluri?into?the?water?by?moribund?fish?just?prior?to?death?or?during?
decomposition?of?moribund?fish?(Wise?et?al.?1997).?Wise?et?al.?(1997)?exposed?channel?catfish?fingerlings?to?E.?ictaluriin?aquaria?and?monitored?the?number?ofE.?ictaluri?cels?
in?water?over?a?216?h?period.?The?concentration?ofE.?ictaluri?in?the?water?significantly?increased?after?48?h?and?this?increase?coincided?with?the?onset?of?mortalities.?Widespread?
use?of?the?multiple?batch?system?by?channel?catfish?producers?may?be?contributing?to?the?continued?spread?of?ESC?(Tucker?and?Robinson?1990).??In?multiple?batch?systems,?na?ve?
fish?are?often?mixed?with?carier?fish.?In?addition,?fish?farmers?may?unintentionally?facilitate?transmision?by?pasing?efluent?from?one?pond?to?another?or?using?seines?that?
had?previously?ben?used?in?ponds?with?infected?animals?without?disinfection?or?drying.?Other?animals?may?facilitate?transmision?by?serving?as?vectors?(Tucker?and?
Robinson?1990).?Taylor?(1992)?performed?intestinal?and?rectal?smears?of?snowy?egrets?Egretta?thula,?great?egretsCasmerodius?albus,?great?blue?herons?Ardea?herodias,?and?
double?crested?cormorants?Phalacrocorax?auritus?and?tested?for?the?presence?ofE.
9?
ictaluri?using?an?indirect?fluorescent?antibody?(IFA)?test.?The?bacterium?was?detected?in?53%?of?sampled?birds.?Results?of?a?study?conducted?by?Waterstrat?et?al.?(1999),?however,?
suggested?that?birds?play?a?limited?role?inE.?ictaluri?transmision.?Over?a?4?d?period,?great?blue?herons?A.?herodias?were?fed?channel?catfish?obtained?from?a?pond?experiencing?
an?outbreak?of?ESC?and?injected?intraperitoneally?with?live?E.?ictaluri.??During?the?feeding?period,?fecal?samples,?throat?and?rectal?swabs,?and?feather?samples?were?
colected.?At?the?end?of?the?4?d?feeding?period,?birds?were?euthenized?and?gastrointestinal?tracts?were?sampled.??Selective?media?was?used?to?attempt?to?recover?the?
bacterium?from?the?samples,?and?an?IFA?test?was?used?to?attempt?to?detect?the?bacterium.?The?bacterium?was?detected?using?IFA,?but?the?researchers?failed?to?recover?viable?E.?
ictaluri.?In?the?same?study,?Waterstrat?et?al.?(1999)?determined?the?body?temperature?of?great?blue?herons?to?be?40.2?C,?and?then?attempted?to?growE.?ictaluri?at?24?C?after?
incubating?the?cultures?at?40?C?for?either?5?h?or?8?h.?No?growth?was?observed?in?the?cultures.?The?failure?to?recover?viable?E.?ictaluri?from?samples?and?the?failure?to?grow?
the?bacterium?at?40?C?led?the?authors?to?conclude?that?great?blue?herons?probably?play?a?limited?role?inE.?ictaluritransmision.?In?support?of?their?findings,?Waterstrat?et?al.?
(1999)?pointed?out?that,?although?Taylor?(1992)?detectedE.?ictaluri?in?53%?of?sampled?birds,?viable?E.?ictaluri?was?recovered?from?only?two?of?the?samples?taken?over?the?three??
year?study?period.?E.?ictaluri?can?enter?channel?catfish?via?the?olfactory?mucosa,?intestine,?or?gills.?
Miyazaki?and?Plumb?(1985)?observed?inflammation?in?olfactory?organs?of?channel?catfish?naturally?infected?withE.?ictaluri.?Newton?et?al.?(1989)?observed?cutaneous?
hemorhaging?and?enteritis?4?d?after?experimentally?inducing?E.?ictaluri?infection?by
10?
immersion.?Meningoencephalitis?involving?the?olfactory?bulbs?and?dorsocranial?ulceration?were?observed?3?4?weeks?post?exposure.?Morison?and?Plumb?(1994)?
observed?damage?to?cilia?on?olfactory?mucosal?surfaces?of?channel?catfish?1?h?post??exposure?to?E.?ictaluri.??Baldwin?and?Newton?(1993)?showed?that?E.?ictaluri?can?cros?
intestinal?mucosa?0.25?h?post?injection?of?the?gut.?Nusbaum?and?Morison?(1996)?immersed?channel?catfish?in?heat?kiled?radiolabeled?E.?ictaluri?and?were?able?to?detect?
the?bacterium?in?or?on?the?gills?within?2?h?of?exposure.?Like?some?other?members?of?Enterobacteriacae,?such?as?Salmonella?sp.?and?
Yersinia?sp.,?E.?ictaluri?has?been?shown?to?be?an?intracellular?pathogen,?surviving?and?reproducing?within?neutrophils?and?macrophages?of?channel?catfish.?Miyazaki?and?
Plumb?(1985)?examined?moribund?fish?with?natural?infections?ofE.?ictaluri.?The?researchers?observed?E.?ictaluricels?in?the?proces?of?dividing?within?neutrophils,?as?
wel?as?neutrophils?having?died?from?extensive?multiplication?of?the?bacterium.?Morison?and?Plumb?(1994)?observed?phagocytic?cels?migrating?through?olfactory?epithelium?to?
phagocytize?E.?ictaluri,?but?failing?to?destroy?the?bacterium.?Baldwin?and?Newton?(1993)?experimentally?infected?channel?catfish?intragastrically?and?later?examined?tisues?by?
electron?microscopy.?Cytoplasmic?vacuoles?of?macrophages?were?found?to?containE.?ictaluri,?with?the?number?of?cells?present?increasing?over?time.?The?researchers?also?
observed?E.?ictaluri?cels?in?the?proces?of?dividing?within?cytoplasmic?vacuoles?of?macrophages.?Shotts?et?al.?(1986)?observed?lesions?in?channel?catfish?resulting?from?
exposure?to?E.?ictuluri?and?noted?macrophages?containing?bacteria?in?every?tisue?examined.?The?authors?suggested?that?macrophages?might?play?an?important?role?in?
spreading?the?bacterium?throughout?the?host.
11?
Extracelular?products?may?contribute?to?the?ability?ofE.?ictalurito?cause?disease?in?channel?catfish.?Stanley?et?al.?(1994)?found?clear?diferences?between?extracelular?
products?produced?by?virulent?strains?ofE.?ictaluri?and?those?produced?by?avirulent?(attenuated)?strains.?Scanning?electron?microscopy?and?transmision?electron?microscopy?
both?showed?virulent?strains?to?have?a?greater?amount?of?capsular?material.?Congo?red,?a?dye?that?binds?to?proteins,?was?used?to?show?that?virulent?strains?posess?more?surface?
proteins?than?avirulent?strains.?A?plate?assay?used?to?evaluate?chondrotinase?activity?revealed?virulent?strains?to?have?greater?ability?to?degrade?chondrotin?sulfate?than?
avirulent?strains.?Stanley?et?al.?(1994)?suggested?that?the?relatively?greater?amount?of?capsular?material?and?surface?proteins?posessed?by?virulent?strains?may?contribute?to?
their?ability?to?attach?to?macrophages?and?to?survive?in?the?acidic?environment?within?macrophages.?The?authors?also?suggested?that?the?greater?ability?of?virulent?strains?to?
degrade?chondroitin?sulfate,?an?abundant?compound?in?cartilage,?may?alow?them?to?escape?host?localization?and?facilitate?their?spread?throughout?the?host.?Shotts?et?al.?
(1986)?suggested?that?chondroitinase?activity?may?be?responsible?for?the?hole?in?the?head?lesion?characteristic?of?ESC.
Imune?System?of?Channel?Catfish?All?multicellular?organisms?poses?some?form?of?innate?imunity,?and?adaptive?
immune?mechanisms?first?evolved?in?jawed?vertebrates?(Abbas?and?Lichtman?2003d).?Therefore,?channel?catfish?posess?an?innate?and?an?adaptive?immune?system.?Innate?
immune?mechanisms?are?nonspecific,?meaning?they?recognize?structures?shared?by?groups?of?organisms,?but?cannot?distinguish?small?diferences?among?microbes.?Adaptive?
immune?mechanisms?are?specific,?meaning?they?can?distinguish?among?diferent
12?
microbes?by?recognizing?antigens?specific?to?those?microbes.?Adaptive?immune?mechanisms?also?show?memory,?meaning?that?subsequent?exposures?to?a?particular?
antigen?elicit?faster?and?stronger?responses?than?the?primary?exposure,?whereas?innate?immune?mechanisms?do?not?have?this?capability.?After?a?host?encounters?an?antigen?for?
the?first?time,?it?passes?from?a?na?ve?status?to?an?immune?status,?with?regard?to?that?particular?antigen?(Abbas?and?Lichtman?2003d).?
The?innate?immune?system?of?channel?catfish?consists?of?physical?barriers,?phagocytic?cells,?nonspecific?cytoxic?cels,?and?the?complement?system.?Physical?barriers?
include?mucous?and?skin.?Goblet?cels?in?the?skin,?gills,?and?gastrointestinal?tract?secrete?mucous,?which?prevents?colonization?of?these?organs?by?microbes.?The?skin?prevents?
microbes?from?directly?entering?the?blod?stream.?Phagocytic?cells?are?cable?of?adhering?to,?engulfing,?and?destroying?microbes?(Moore?and?Hawke?2004).?Macrophages?and?
neutrophils?are?the?primary?phagocytic?cels?in?channel?catfish?(Ainsworth?1992??Finco??Kent?and?Thune?1987).?Nonspecific?cytoxic?cells?are?similar?to?mammalian?natural?killer?
(NK)?cels?in?that?they?are?nonspecific,?are?not?capable?of?phagocytosis,?participate?in?mediating?the?lysis?of?virus?infected?cels,?protozoan?parasites?and?tumor?target?cels,?and?
require?cel?contact?with?the?target?cel?to?mediate?lysis?(Evans?et?al.?1984).?In?mammalian?systems,?complement?is?a?system?composed?of?serum?and?mucous?
proteins,?designated?C1?through?C9,?that?when?activated?interact?in?a?sequential?cascade?of?protelytic?steps.?Activation?of?complement?can?result?in?formation?of?the?membrane?
attack?complex,?which?lyses?cel?membranes??complement?mediated?opsonization??or?complement?mediated?inflammation.?Complement?mediated?opsonization?is?the?proces?
in?which?complement?fragments?coat?microbes,?thereby?facilitating?phagocytosis?of
13?
microbes.?Complement?mediated?inflammation?describes?the?proces?in?which?complement?fragments?trigger?an?inflammatory?response?vasodilation,?increased?
vascular?permeability,?and?migration?of?white?blod?cels?to?the?site?of?infection.?Complement?can?be?activated?by?either?the?alternative?or?classical?pathway.?Activation?of?
the?alternative?pathway?occurs?nonspecifically,?whereas?activation?of?the?classical?pathway?requires?specific?antibodies?(Abbas?and?Lichtman?2003c).?
Complement?activation?pathways?in?fish?appear?to?be?similar?to?those?in?mammals?(Holand?and?Lambris?2002).?The?alternative?and?clasical?pathways?can?activate?
complement?in?channel?catfish?(Ourth?and?Wilson?1982).?The?effector?mechanisms?of?complement?in?fish?also?appear?to?be?similar?to?those?in?mamals.?Fish?complement?has?
been?shown?to?function?in?lysis?of?foreign?cells,?opsonization,?and?inflammation.?Furthermore,?complement?proteins?in?fish?appear?to?be?similar?to?those?in?mammals,?but?
tend?to?be?more?diverse.??For?example,?C3?has?been?shown?to?occur?in?five?different?forms?(Holand?and?Lambris?2002).?In?channel?catfish?a?C1q?type?protein?(Dodds?and?
Petry?1993)?and?proteins?analogous?to?mammalian?C3?and?C4?have?ben?isolated?(Jenkins?and?Ourth?1991??Holand?and?Lambris?2002).?
As?in?mamals,?two?types?of?adaptive?immunity?exist?in?fish:?humoral?and?cel??mediated?(Klesius?1992b).?Humoral?immunity?consists?of?B?cels?defined?as?those?
lymphocytes?(antigen?specific?leukocytes)?that?express?immunoglobulin?(antibodies)?on?their?surface?and?secrete?specific?antibody?in?response?to?antigenic?stimulation?(Kaatari?
1992).?Cel?mediated?imunity?consists?of?T?like?cels,?which,?despite?lacking?cell?surface?markers?as?mammalian?T?cels?posess?have?functions?similar?to?mammalian?
T?helper?and?cytolytic?T?cels?(CTLs)?(Chilmonczyk?1992).?B?cels?are?formed?in?the
14?
anterior?kidneys?of?fish?(Kaattari?1992).?Upon?activation,?B?cels?either?diferentiate?into?memory?cells?or?plasma?cells.?Plasma?cells?no?longer?express?membrane?bound?
immunoglobulin,?but?instead?secrete?antibodies?(Abbas?and?Lichtman?2003c).?Activation?of?B?cels?occurs?when?they?specifically?recognize?one?of?two?types?of?antigens:?T??
independent?or?T?dependent.?T?independent?antigens,?which?are?generally?polysaccharides,?do?not?require?the?assistance?of?T?helper?cels?to?activate?B?cels,?but?T??
dependent?antigens,?which?tend?to?be?proteins,?do?require?the?assistance?of?T?helper?cels?(Kaatari?and?Piganelli?1996).?
Imunization?of?fish?triggers?B?cells?to?produce?specific?antibodies?that?appear?in?blod,?bile,?and?mucous?(Wilson?and?Warr?1992).?Studies?have?shown?that?E.?ictaluri??
specific?serum?antibodies?are?indicative?of?prior?exposure?to?E.?ictaluri(Klesius?1992b??Vinitnantharat?and?Plumb?1993??Antonio?and?Hedrick?1994).?Serological?detection?ofE.?
ictaluri?specific?antibodies?have?been?used?in?diagnosing?E.?ictaluri?infections?(Klesius?et?al.?1991).?The?presence?of?serum?antibodies?after?immunization?has?been?confirmed?in?
numerous?studies.?Plumb?and?Vinitnantharat?(1993)?vaccinated?fry?against?ESC?by?immersion?in?bacterin?(killed?bacteria),?stocked?the?fish?into?ponds,?and?ofered?an?oral?
bacterin?boster?to?a?portion?of?the?previously?vaccinated?fry.?Antibody?titers?were?significantly?higher?in?vaccinated?fish?than?in?control?fish.?Thune?et?al.?(1997)?vaccinated?
channel?catfish?against?ESC?by?immersion?bacterin?or?imersion?bacterin?folowed?by?an?oral?bacterin?boster?and?later?assayed?serum?antibodies.?Both?vaccination?regimes?
resulted?in?antibody?titers?significantly?higher?than?those?observed?in?control?fish.?Shoemaker?and?Klesius?(1997)?vaccinated?juvenile?channel?catfish?by?immersion?bacterin
15?
or?oral?bacterin?and?later?assayed?serum?antibodies.?Antibody?titers?were?significantly?higher?in?fish?vaccinated?by?immersion?bacterin?than?in?control?fish.?
Increases?in?antigen?concentration,?water?temperature?or?antigen?exposure?time?have?been?shown?to?increase?the?magnitude?of?the?primary?immune?response.?
Vinitnantharat?and?Plumb?(1992)?injected?channel?catfish?intraperitoneally?with?killed?E.?ictaluri?at?varying?doses.?Fish?exposed?to?2.0?x?10?4?cels/fish?had?lower?antibody?titers?
than?fish?exposed?to?2.0?x?10?6?or?2.0?x?10?8?cels/fish.?The?researchers?injected?a?second?group?of?channel?catfish?intraperitoneally?with?an?extract?ofE.?ictaluri?at?varying?
temperatures.?Fish?immunized?at?20?C?had?lower?antibody?titers?than?fish?immunized?at?25?C?or?30?C.?The?researchers?immersed?a?third?group?of?channel?catfish?in?an?extract?of?
E.?ictaluri?for?varying?exposure?times.?Fish?exposed?to?antigen?for?30?min?or?8?h?had?higher?antibody?titers?than?fishexposed?for?2?min?or?5?min.?
As?in?mamals,?fish?antibodies?function?by?neutralization,?agglutination,?opsonization,?and?complement?activation?(Wilson?and?Warr?1992).?Neutralization?is?the?
proces?in?which?specific?antibodies?bind?to?antigen?receptors,?thereby?preventing?attachment?of?antigens?to?cel?targets.?Agglutination?is?the?proces?in?which?antibodies?
cros?link?antigens?on?multiple?cell?surfaces?causing?cells?to?clump?together?and?thereby?facilitating?removal?of?bacteria?by?macrophages.?As?withcomplement,?
opsonization?is?the?proces?in?which?antibodies?coat?the?surface?of?antigens,?facilitating?phagocytosis?of?antigens.?Antibodies?can?activate?complement?via?the?classical?pathway?
(Abbas?and?Lichtman?2003c).?Unlike?mamalian?antibodies,?which?occur?in?five?diferent?isotypes,?only?one?
isotype?is?present?in?fish.?Because?of?its?resemblance?in?size,?structure,?gene?organization
16?
and?physiochemical?properties?to?mammalian?IgM,?the?isotype?present?in?fish?is?referred?to?as?IgM?like.??As?in?mammals,?the?monomeric?form?of?the?molecule?has?two?binding?
sites.?However,?the?monomers?tend?to?associate?to?form?tetramers,?so?that?each?tetrameric?association?has?eight?binding?sites?(Wilson?and?War?1992).?
In?mammalian?systems,?a?secondary?exposure?to?an?antigen?results?in?faster?and?greater?increases?in?specific?antibodies,?isotype?switching?(generation?of?isotypes?other?
than?IgM),?and?afinity?maturation?(generation?of?antibodies?with?increasingly?higher?afinities?over?time)?(Abbas?and?Lichtman?2003a).?The?secondary?response?of?fish?is?
similar?to?that?of?mammals,?in?that?faster?and?greater?increases?in?specific?antibodies?are?seen,?but?is?disimilar?to?that?of?mammals,?in?that?isotype?switching?and?afinity?
maturation?are?not?seen?(Wilson?and?Warr?1992).?Studies?have?shown?channel?catfish?mount?a?secondary?humoral?immune?response?to?E.?ictaluri?greater?than?the?primary?
response.??Vinitnantharat?and?Plumb?(1992)?injected?channel?catfish?intraperitoneally?with?extracts?ofE.?ictaluri?and?thereafter?monitored?serum?antibody?titers?on?a?weekly?
basis.?Antibody?titers?rose?for?4?weeks?and?then?declined.?Folowing?a?boster?injection?administered?6?weeks?post?primary?vaccination,?antibody?titers?reached?levels?nearly?
twice?those?reached?during?the?primary?response.??Petrie?Hanson?and?Ainsworth?(1999)?vaccinated?channel?catfish?fry?4?weeks?post?hatch?with?live?E.?ictaluri?and?reexposed?
them?to?the?bacterium?at?8?weeks?post?hatch.?Antibodies?were?assayed?post?primary?and?post?secondary?exposure.??Antibody?titers?were?significantly?higher?post?secondary?
exposure?than?post?primary?exposure.?Although?Vinitnantharat?and?Plumb?(1992)?and?Petrie?Hanson?and?Ainsworth?
(1999)?observed?greater?secondary?responses,?it?should?be?noted?that?Klesius?and?Sealy
17?
(1995)?observed?a?lesser?secondary?response.??However,?the?authors?did?not?attribute?the?leser?secondary?response?to?lack?of?immunological?memory.?Klesius?and?Sealy?(1995)?
immersed?channel?catfish?in?virulent?E.?ictalurito?achieve?primary?or?secondary?responses?and?thereafter?monitored?serum?antibody?titers?on?a?weekly?basis.??During?both?
the?primary?and?secondary?exposures,?significant?specific?antibody?titers?were?observed,?although?titers?were?higher?during?the?primary?response.?The?researchers?ofered?four?
explanations?for?why?they?may?have?observed?lower?antibody?production?during?the?secondary?response?than?in?the?primary?response:?antibodies?may?have?functioned?in?
opsonization??antibodies?may?have?ben?trapped?in?immune?complexes??fish?may?have?down?regulated?a?strong?immune?response??or?immersion?may?simply?have?not?resulted?in?
a?strong?memory?response.?In?most?cases,?a?specific?immune?response?involves?both?humoral?and?cel??
mediated?imune?mechanisms?(Moore?and?Hawke?2004).?B?cels?can?only?become?activated?in?the?absence?of?a?cell?mediated?response?if?the?antigen?is?T?independent??
additionally,?T?independent?antigens?do?not?lead?to?immunological?memory?in?B?cels.?Imunological?memory?develops?only?when?B?cells?are?activated?by?T?dependent?
antigens?in?conjunction?with?T?helper?cels?(Arkoosh?and?Kaatari?1991).?T?cels,?both?T??helper?and?CTLs,?arise?in?the?thymus?(Chilmonczyk?1992),?and?when?activated?by?
specifically?recognizing?a?particular?antigen?they?either?diferentiate?into?effector?T?cels?or?memory?cells?(Abbas?and?Lichtman?2003a).?Efector?T?helper?cels?secrete?
cytokines?soluble?messenger?proteins?that?are?involved?in?activating?B?cells,?macrophages,?and?neutrophils.?Efector?CTLs?are?involved?in?destroying?altered?host?
cels?(Manning?and?Nakanishi?1996).
18?
For?T?cels?to?recognize?antigens?in?mammalian?systems,?antigens?must?be?displayed?by?major?histocompatability?(MHC)?molecules.?MHC?molecules?are?present?
on?host?cels?referred?to?as?target?cels?and?on?B?cels,?macrophages?and?neutrophils??referred?to?as?antigen?presenting?cells?(APCs).?MHC?clas?I?molecules?are?present?on?
target?cels?and?display?antigens?to?CTLs.?MHC?class?I?molecules?are?present?only?on?APCs?and?display?antigens?to?T?helper?cels.?APCs?must?proces,?or?break?antigens?up?
into?short?peptides,?before?displaying?them?on?MHC?molecules?(Abbas?and?Lichtman?2003b).?Antigen?procesing?and?presentation?in?teleosts?have?been?shown?to?be?similar?to?
that?of?mamals?(Valejo?et?al.?1992).?Factors?Affecting?the?Imune?System?
Nutrition?significantly?influences?the?imune?system?of?channel?catfish.?Micronutrients?known?to?improve?resistance?to?disease?include?vitamins?C,?B6,?E,?and?A?
and?the?minerals?iron?and?fluoride?(Blazer?1992).?Wise?et?al.?(1993)?fed?channel?catfish?fingerlings?diets?containing?0,?60,?or?2500?mg?vitamin?E/kg,?vaccinated?them?by?
immersion?in?a?bacterin?folowed?by?an?oral?boster,?and?later?analyzed?for?macrophage?activity.?The?ability?of?macrophages?to?phagocytize?virulent?E.?ictaluri?was?enhanced?in?
both?vaccinates?and?non?vaccinates?fed?either?60?or?2500?mg?vitamin?E/kg.?Sealey?et?al.?(1997)?found?that?channel?catfish?deficient?in?dietary?iron?were?more?susceptible?to?
infection?byE.?ictaluri.?Paripatananont?and?Lovell?(1995)?fed?channel?catfish?diets?supplemented?with?varying?amounts?of?zinc?and?later?chalenged?them?withE.?ictaluri.?
Fish?receiving?no?zinc?in?their?diets?showed?100%?mortality,?but?those?fish?receiving?an?adequate?amount?of?zinc?showed?25?30%?mortality.?Duncan?and?Lovell?(1994)?fed?
channel?catfishdiets?containing?0,?0.4,?or?4.0?mg?folic?acid/kg?with?0,?20,?or?200?mg
19?
vitamin?C/kg?in?a?factorial?design?in?aquaria?and?later?chalenged?them?withE.?ictaluri.?Maximum?survival?was?observed?in?fish?fed?diets?containing?0.4?or?4.0?mg?folic?acid/kg?
in?combination?with?the?high?level?of?vitamin?C.?However,?improved?survival?was?not?observed?in?fish?fed?diets?supplemented?with?vitamin?C?or?folic?acid?alone.?
Certain?fatty?acids?may?play?a?role?in?disease?resistance?(Blazer?1992).?Li?et?al.?(1994)?fed?channel?catfish?diets?supplemented?with?2%?catfish?ofal?oil,?beef?talow,?or?
menhaden?oil?and?challenged?them?withE.?ictaluri.?Fish?fed?the?diet?supplemented?with?menhaden?oil?showed?increased?susceptibility?to?E.?ictaluri,?but?those?fed?the?other?diets?
did?not?show?increased?susceptibility.?Sheldon?and?Blazer?(1991)?vaccinated?channel?catfish?by?immersion?in?killed?E.?ictaluri,?ofered?vaccinated?and?non?vaccinated?fish?
diets?of?varying?lipid?content,?and?later?isolated?macrophages?from?both?vaccinated?and?non?vaccinated?fish.?Isolated?macrophages?were?exposed?to?E.?ictalurito?assay?
bactericidal?activity.?Bactericidal?killing?of?macrophages?increased?as?the?level?of?n?3?fatty?acids?in?the?diet?increased?in?both?non?vaccinated?and?vaccinated?fish.?
An?indirect?relationship?has?been?shown?to?exist?between?the?longevity?of?circulating?antibodies?and?water?temperature.?Plumb?et?al.?(1986)?assayed?antibodies?60?
d?post?vaccination?in?channel?catfish?vaccinated?by?injection?with?killed?E.?ictaluri?and?held?at?diferent?temperature?regimes.?Higher?antibody?titers?were?observed?in?vaccinated?
fish?held?at?25?C?for?30?d?and?12?C?for?an?additional?30?d?than?vaccinated?fish?held?at?25?C?for?60?d.?Higher?antibody?titers?were?also?observed?in?vaccinated?fish?held?at?25C?for?
5?or?10?d?and?then?held?at?12?C?until?60?d?post?vaccination?than?in?vaccinated?fish?held?at?25?C.
20?
A?rapid?drop?in?water?temperature?from?23?C?to?11?C?has?been?shown?to?result?in?suppression?of?T?helper?cels?and?B?cels.?Bly?and?Clem?(1991)?decreased?the?water?
temperature?in?which?channel?catfish?were?immersed?from?23?C?to?11?C?over?a?24?h?period.?Subsequently,?T?and?B?cels?responses?to?mitogen?and?antibody?production?
folowing?stimulation?by?T?dependent?or?T?independent?antigens?were?assayed.??The?researchers?observed?a?reduction?in?T?and?B?cel?proliferation?in?response?to?mitogen?and?
suppressed?antibody?production?in?response?to?either?T?dependent?or?T?independent?antigen.?
A?rapid?drop?in?water?temperature?from?22?C?to?17?C?has?also?beenshown?to?result?in?suppresion?of?T?helper?cels.?Clem?et?al.?(1984)?cultured?B?cels?and?T?helper?
cels?isolated?from?channel?catfish?acclimated?to?22?C?in?the?presence?of?LPS?(a?B?cell?mitogen)?or?Con?A?(a?T?cel?mitogen)?at?32,?27,?22,?or?17?C.?B?cels?showed?a?significant?
response?to?LPS,?regardless?of?culture?temperature.?T?helper?cells?showed?significant?responses?to?Con?A?at?32?and?27?C,?a?diminished?response?at?22?C,?and?no?response?at?17?
C.?Miller?and?Clem?(1984)?cultured?B?cells?isolated?fromchannel?catfish?acclimated?to?22?C?in?the?presence?of?T?dependent?and?T?independent?antigens?at?32,?27,?22,?or?17?C.?
B?cels?showed?significant?responses?to?T?independent?antigens,?regardless?of?culture?temperature.?However,?B?cels?only?showed?significant?responses?to?T?dependent?
antigens?at?32?and?27?C.?Diminished?B?cell?responses?were?observed?at?22?and?17?C,?with?the?response?at?17?C?much?lower?than?that?at?22?C.?
Although?a?rapid?drop?in?temperature?from?22?C?to?17?C?has?been?shown?to?result?in?suppression?of?T?helper?cels,?acclimation?of?fish?to?17?C?has?been?shown?to?result?in?
recovery?of?T?helper?cel?function.?Clem?et?al.?(1984)?cultured?T?helper?cels?isolated
21?
from?channel?catfish?acclimated?for?at?least?2?weeks?to?17?C?in?the?presence?of?ConA?at?32,?27,?22,?or?17?C.?T?helper?cels?showed?significant?responses?to?Con?A?at?32,?27,?and?
22?C.?Miler?and?Clem?(1984)?cultured?B?cels?isolated?from?channel?catfish?acclimated?to?17?C?in?the?presence?of?T?dependent?and?T?independent?antigens?at?27,22,?or?17?C.?B??
cels?showed?significant?responses?to?both?T?independent?and?T?dependent?antigens,?regardless?of?culture?temperature.?
Stress?has?been?defined?as?the?response?of?animals?to?changes?or?chalenges?in?their?environments?by?a?variety?of?interlocking?anatomical,?physiological,?biochemical,?
immunological,?and?behavioral?adaptation?mechanisms?(Ewbank?1989).?Events?triggering?a?stress?response?include:?osmotic?and?ionic?changes,?polutants,?crowding,?
handling,?grading,?hauling,?and?other?aquaculture?practices?(Eddy?1981).?The?physiological?changes?associated?with?stress?can?be?divided?into?three?response?phases:?
primary,?secondary?and?tertiary.?During?the?primary?response?phase,?plasma?concentrations?of?corticosteriods?and?catecholamines?rise,?leading?to?the?secondary?phase?
which?afects?metabolic?and?osmoregulatory?functions.?Changes?in?plasma?glucose?levels?and?fatty?acids?also?accompany?these?disturbances?(Mazeaud?et?al.?1977).?During?the?
tertiary?phases,?behavioral?changes,?decreased?growthand?immunosuppression?can?be?observed?(Wedemeyer?and?McLeay?1981).?The?tertiary?efect?of?immunosuppression?
results?from?the?ability?of?corticosteriods,?such?as?cortisol,?to?inhibit?leukocyte?migration,?lymphocyte?effector?mechanisms,?inflammation,?and?cytokine?production?(Kennedy??
Stoskopf?1993).?Stress?caused?by?transport?and?handling?has?been?shown?to?result?in?
immunosuppression?in?channel?catfish.?In?a?study?by?Klesius?(1992b)?channel?catfish
22?
fingerlings?known?to?have?sufered?from?clinical?ESC?wereto?be?used?as?control?fish,?but?approximately?one?third?of?the?fish?developed?clinical?signs?associated?with?ESC?and?died?
3?d?after?moving?them?into?the?laboratory.?Klesius?determined?that?a?combination?of?ESC?and?transport?induced?stress?caused?the?mortalities.?Wise?et?al.?(1993)?subjected?channel?
catfish?to?a?30?min?standardized?confinement?treatment?intended?to?simulate?current?handling?practices?used?in?channel?catfish?culture?and?60?min?later?to?static?immersion?
inE.?ictaluri?for?24?or?48?h.?Unstressed?fish?were?also?subjected?to?static?immersion?for?24?or?48?h.?Mortalities?were?significantly?higher?in?stressed?fish?and?those?exposed?to?E.?
ictaluri?for?48?h.?Ciembor?et?al.?(1995)?exposed?channel?catfish?sac?fry?to?E.?ictaluri?by?bath?immersion?with?stress?(induced?by?handling?and?netting)?or?bath?immersion?without?
stress.?Three?weeks?after?exposure,?52.7%?of?fish?from?the?stressed?group?tested?positive?for?E.?ictaluri,?while?only?15.7%?of?fish?from?the?unstressed?group?tested?positive.?
Stress?caused?by?low?disolved?oxygen?or?rapid?changes?in?water?temperature?has?also?been?shown?to?result?in?immunosuppression?in?channel?catfish.?Mqolomba?and?
Plumb?(1992)?experimentally?infected?channel?catfish?withE.?ictaluriand?subjected?them?to?one?of?three?treatments:?25?C?with?aeration,?25?C?with?no?aeration,?and?variable?
temperature?(18?23?C)?with?no?aeration.?Concentrations?ofE.?ictaluri?within?tisues?were?significantly?higher?in?fish?held?in?25?C?water?with?no?aeration?and?in?variable?
temperature?water?with?no?aeration?than?in?fish?held?in?25?C?water?with?aeration.?Imunosuppression?asociated?with?temperature?fluctuations?may?contribute?to?
the?seasonality?of?ESC?outbreaks.?Outbreaks?of?ESC?tend?to?occur?in?the?late?spring?and?early?fall,?when?temperature?fluctuations?most?often?occur.?Temperature?fluctuations?
might?compromise?the?immune?system?of?carier?fish,?leading?to?development?of?ESC?in
23?
these?fish?and?transmision?to?susceptible?fish?(Klesius?1992a).?Mention?has?already?ben?made?of?the?fact?that?spring?and?fall?temperatures?in?the?southeastern?United?States?
corespond?to?the?optimal?temperatures?for?ESC?outbreaks?(Tucker?and?Robinson?1990).?Another?posible?reason?for?the?seasonality?of?ESC?may?be?that?high?feed?alotments?
ofered?during?the?fall?can?lead?to?low?oxygen?and?poor?water?quality,?which?in?turn?lead?to?stress?and?immunosuppression.?
Imune?Response?of?Channel?Catfish?to?E.?ictaluri?A?study?by?Ainsworth?and?Dexiang?(1990)?suggested?that?complement?may?play?a?
role?in?protecting?against?ESC?by?opsonizing?E.?ictaluri.?The?researchers?performed?phagocytic?assays?by?mixing?neutrophils,?E.?ictaluri,?and?normal?or?inactivated?serum.?
The?authors?calculated?percent?phagocytosis?by?counting?the?number?of?neutrophils?containing?at?least?one?bacterium?out?of?100?neutrophils?counted.?Percent?phagocytosis?
of?the?normal?serum?treatment?(63.31%)?was?significantly?higher?than?that?of?the?inactivated?serum?treatment?(8.63%).?The?authors?suggested?that?opsonization?by?
specific?antibodies?or?complement?may?have?accounted?for?the?diference,?but?pointed?out?that?only?10%?of?fish?in?the?pond?in?which?experimental?fish?were?sampled?had?detectable?
titers?of?antibodies?specific?to?E.?ictaluri.?While?complement?may?play?a?role?in?protecting?against?ESC?by?opsonizing?E.?
ictaluri,?a?study?by?Ourth?and?Bachinski?(1987)?indicated?that?it?is?unlikely?that?complement?protects?against?ESC?by?lysing?E.?ictaluri.?Knowing?that?sialic?acid?
hindered?the?binding?of?complement?factor?B?of?the?alternative?complement?pathway?(ACP),?the?researchers?determined?sialic?acid?content?and?examined?bactericidal?activity?
by?channel?catfish?serum?incubated?at?30?C?for?1?h?on?fifteen?Gram?negative?fish
24?
pathogens,?including?E.?ictaluri,?and?non?pathogens.?Ourth?and?Bachinski?(1987)?found?that?non?pathogens?contained?90??g?or?les?per?100?mg?of?dried?bacteria?and?had?100%?
bactericidal?activity?produced?against?them.?Pathogens?contained?greater?than?90??g?per?100?mg?of?dried?bacteria?had?0?13%?bactericidal?activity?produced?against?them.?The?
sialic?acid?content?ofE.?ictaluri?was?123??g?per?100?mg?of?dried?bacteria?and?had?0%?bactericidal?activity?produced?against?it.?Thus,?sialic?acid?may?be?a?mechanism?of?
resistance?ofE.?ictaluri?against?the?ACP.?A?few?studies?have?shown?a?positive?corelation?betweenE.?ictaluri?specific?
antibody?titers?and?protection?against?ESC.?Vinitnantharat?and?Plumb?(1993)?took?serum?samples?from?channel?catfish?that?had?survived?a?naturalE.?ictaluri?infection?and?fin??
clipped?each?fish?to?allow?for?later?identification.?After?assaying?serum?antibodies,?fish?were?divided?into?four?groups?based?on?antibody?titers:?negative?(0),?low?(<128),?medium?
(256?512),?and?high?(>1024).?Treatment?groups?were?challenged?by?intraperitoneal?injection?and?mortality?rates?were?compared.?A?strong?positive?corelation?(regression?
coefficient?=?0.95)?was?observed?between?mortality?rate?and?antibody?titer.?The?researchers?determined?that?antibody?titers?greater?than?1:256?were?capable?of?providing?
protection.?However,?it?should?be?noted?that?challenge?was?performed?by?intraperitoneal?injection,?which?does?not?ofer?the?best?simulation?of?natural?exposure.?Plumb?and?
Vinitnantharat?(1993)?vaccinated?fry?by?immersion?bacterin,?stocked?them?into?ponds,?and?ofered?an?oral?bacterin?boster?to?a?portion?of?the?previously?vaccinated?fry.?Some?
fish?were?removed?before?harvest?and?subjected?to?a?laboratory?challenge.??Survival?and?antibody?titers?were?significantly?higher?in?fish?vaccinated?by?either?regime?than?in?
control?fish.
25?
Humoral?immunity?may?play?a?role?in?protecting?against?ESC?by?opsonizing?E.?ictaluri.?Waterstrat?et?al.?(1991)?performed?phagocytic,?bactericidal,?and?
chemiluminescent?assays?to?determine?the?in?vitroresponses?of?channel?catfish?neutrophils?to?E.?ictaluri.?Although?phagocytosis?ofE.?ictaluriby?neutrophils?was?made?
evident?by?light?and?electron?microscopy,?the?number?of?intracelular?bacteria?remained?either?constant?or?increased?over?the?time?period.?However,?a?control?assay?containing?
a?mixture?of?cels,?bacteria,?and?immune?catfish?serum?exhibited?a?decline?in?viable?E.?ictaluri?cels,?indicating?that?serum?components?or?serum?components?working?in?
conjunction?with?neutrophils?had?bactericidal?activity.?Using?an?oxidative?microbiocidal?activity?as?an?indicator?of?the?chemiluminescent?response?of?neutrophils?to?E.?ictaluri?
opsonized?with?immune?serum?was?twice?the?response?of?neutrophils?to?E.?ictaluri?opsonized?with?non?immune?serum.?The?authors?suggested?that,?although?intracelular?
killing?was?not?observed,?the?extracelular?killing?ofE.?ictaluriobserved?in?the?control?assay?may?have?ben?due?to?serum?components,?extracellular?products?liberated?from?the?
neutrophil,?or?by?a?combination?of?both?celular?and?serum?factors.?The?increased?chemiluminescent?response?observed?with?immune?serum?ay?have?been?due?to?
increased?extracelular?killing.?Although?a?few?studies?have?showed?a?positive?corelation?betweenE.?ictaluri??
specific?antibody?titers?and?protection?against?ESC,?a?majority?have?showed?a?lack?of?or?negative?corelation?between?antibody?titer?and?survival,?indicating?that?humoral?
immunity?is?probably?not?the?only?arm?of?adaptive?immunity?acting?to?protect?against?ESC.?Klesius?and?Sealy?(1995)?passively?immunized?na?ve?channel?catfish?withserum?
from?immune?fish?and?chalenged?them.?Although?antibody?titers?were?confirmed?to?be
26?
significantly?higher?in?pasively?immunized?fish?than?in?control?fish,?protection?was?not?observed.?The?researchers?suggested?that?antibody?titers?may?not?have?beenas?high?as?
those?reached?in?the?study?by?Vinitnantharat?and?Plumb?(1993),?which?might?account?for?the?disparity?between?the?two?studies.?Alternatively,?they?point?out?that?antibodies?may?
be?more?important?in?providing?protection?when?fish?are?challenged?intraperitoneally,?as?in?Vinitnantharat?and?Plumb?s?study?(1993),?than?when?fish?are?challenged?by?immersion.?
In?a?laboratory?experiment,?Plumb?et?al.?(1986)?found?that?vaccination?by?immersion?in?sonicated?bacteria?ofered?greater?protection?than?that?found?in?groups?
vaccinated?by?other?methods,?even?though?it?resulted?in?the?lowest?antibody?titers.??In?a?field?experiment,?Plumb?et?al.?(1986)?vaccinated?fish?by?immersion?or?by?intraperitoneal?
injection,?with?either?sonicated?or?whole?cellE.?ictaluri.?Vaccinated?fish?were?grown?out?in?ponds?and?challenged?under?laboratory?conditions?at?harvest.?Vaccination?by?
immersion?in?sonicated?bacteria?ofered?the?greatest?protection,?even?though?it?resulted?in?the?lowest?antibody?titers.?Plumb?et?al.?(1994)?stocked?fry?into?earthen?ponds?at?low,?
intermediate,?or?high?densities,?and?later?vaccinated?them?orally.?The?researchers?found?that?survival?within?a?vaccinated?or?non?vaccinated?regime?increased?as?stocking?density?
decreased.?However,?antibody?titers?were?low?in?all?treatments,?regardles?of?stocking?density?or?vaccination?regime.?
In?a?laboratory?study,?Wolters?et?al.?(1996)?vaccinated?channel,?blue,?and?hybrid?catfish?by?immersion?or?injection?with?live?E.?ictaluri?and?challenged?them.??When?serum?
antibodies?were?later?assayed,?antibody?titers?were?negatively?corelated?with?survival,?regardless?of?the?challenge?method.?Thune?et?al.?(1997)?performed?a?laboratory?study?to?
evaluate?the?ability?of?vaccination?by?immersion?in?bacterin,?immersion?in?bacterin
27?
folowed?by?an?oral?boster,?or?injection?to?induce?a?humoral?immune?response.?Antibody?titers?were?monitored?throughout?the?study?by?periodically?taking?serum?
samples.?All?vaccination?regimes?resulted?in?measurable?antibody?titers??however,?a?large?percentage?of?seropositive?fish?in?a?group?did?not?corelate?with?increased?survival?in?that?
group.?The?intracelular?nature?ofE.?ictaluri?suggests?that?cel?mediated?immunity,?
working?in?conjunction?with?innate?and?humoral?immunity,?would?be?important?in?providing?protection.?Cell?mediated?imunity?is?the?major?protective?immune?response?
against?most?intracelular?pathogens?(Abbas?and?Lichtman?2003e).?Humoral?immunity?acting?alone?was?shown?to?fail?in?protecting?mice?and?catle?against?the?intracelular?
pathogen,?Brucella?abortus?(Montaraz?and?Winter?1986).?Klesius?(1992a)?pointed?out?that,?just?as?both?the?humoral?and?cell?mediated?components?of?the?adaptive?immune?
system?cooperate?to?provide?protection?in?mammals,?the?same?is?probably?true?in?catfish.?Several?studies?have?shown?that?cel?mediated?imunity?is?important?in?providing?
protection?against?ESC.?Shoemaker?and?Klesius?(1997)?vaccinated?channel?catfish?fingerlings?with?live?E.?ictaluri?and?later?chalenged?them.?Antibody?titers?and?
bactericidal?activity?by?peritoneal?macrophages?were?assayed?folowing?chalenge.?Survival,?antibody?titers,?and?macrophage?bactericidal?activity?were?significantly?higher?
in?vaccinated?fish?than?in?non?vaccinated?fish.?Shoemaker?et?al.?(1997)?isolated?macrophages?from?na?ve?channel?catfish?and?channel?catfish?surviving?exposure?to?live?E.?
ictaluri.?Isolated?macrophages?were?incubated?with?live?E.?ictaluri,?and?macrophage?bactericidal?activity?was?assayed.??Macrophage?bactericidal?activity?was?significantly
28?
higher?in?immune?fish?than?in?naive?fish,?indicating?that?macrophages?isolated?from?immune?fish?had?been?activated?by?T?helper?cells.?
In?a?separate?study,?Shoemaker?et?al.?(1997)?incubated?macrophages?isolated?from?na?ve?or?imune?channel?catfish?in?opsonized?or?unopsonized?E.?ictaluri.?The?
bactericidal?activity?of?macrophages?isolated?from?immune?fish?incubated?in?opsonized?bacteria?was?higher?at?2.5?h?post?exposure?than?that?of?macrophages?incubated?in?
unopsonized?bacteria.??However,?the?bactericidal?activityof?macrophages?isolated?from?na?ve?fish?incubated?in?opsonized?bacteria?was?suppressed?at?1.5,?3,?and?5?h?post??
exposure,?compared?to?that?of?macrophages?incubated?in?unopsonized?bacteria.?Shoemaker?et?al.?(1997)?suggested?that?activation?of?macrophages?occurred?in?immune?
fish,?alowing?the?macrophages?to?engulf?opsonized?bacteria.?Klesius?and?Shoemaker?(1997)?suggested?that,?upon?a?secondary?exposure?to?E.?ictaluri,?antibodies?generated?by?
immune?fish?may?opsonize?the?bacterium,?facilitating?its?engulfment?by?activated?macrophages,?where?the?bacterium?is?then?killed.?
Antonio?and?Hedrick?(1994)?treated?carrier?fish?with?Kenalog,?an?immunosuppressive?drug,?and?reexposed?them?to?E.?ictaluri.?Two?weeks?after?chalenge,?
the?researchers?attempted?to?recover?the?bacterium?and?assayed?serum?antibodies.??Some?fish?died?from?the?reexposure,?despite?having?high?antibody?titers.??In?addition,?the?
researchers?were?able?to?recover?the?bacterium?from?some?surviving?fish,?regardles?of?their?antibody?titers.?Antonio?and?Hedrick?(1994)?concluded?Kenalog?may?have?
suppressed?one?of?the?components?of?cell?mediated?immunity?important?in?providing?protection?against?E.?ictaluri.
29?
Sheldon?and?Blazer?(1991)?vaccinated?channel?catfish?by?intraperitoneal?injection?of?killed?E.?ictaluri,?ofered?vaccinated?and?non?vaccinated?fish?diets?of?varying?lipid?
content,?and?later?isolated?macrophages?from?both?vaccinated?and?non?vaccinated?fish.?Isolated?macrophages?were?exposed?to?E.?ictaluri?at?optimal?and?suboptimal?temperatures?
to?assay?bactericidal?activity.?Imunization?generally?resulted?in?substantial?enhancement?of?bactericidal?activity,?regardless?of?diet?and?temperature.?Phagocytic?
assays?were?also?performed?with?isolated?macrophages?at?optimal?and?suboptimal?temperatures.?Neither?diet?nor?imunization?increased?phagocytosis?at?the?suboptimal?
temperature,?even?though?serum?antibodies?were?high.?The?authors?suggested?that?macrophages?from?immune?fish?showed?increased?bactericidal?activity,?not?because?of?
opsonization?by?antibody,?but?because?macrophages?in?immune?fish?had?undergone?activation.?
Control?Methods?Fish?farmers?have?attempted?to?control?ESC?outbreaks?by?ofering?medicated?feed?
(Tucker?and?Robinson?1990).?Romet???(a?5:1?mixture?of?sulfadimethoxine?and?ormetoprim)?and?Aquaflor???(florfenicol)?are?the?only?antibiotics?approved?by?the?FDA?for?
treating?ESC?(Gaunt?et?al.?2003).?Various?problems?are?inherent?in?treating?with?antibiotics.?Antibiotic?treatment?is?expensive?and?fish?afected?with?ESC?typically?reduce?
their?feeding?activity,?leading?to?antibiotic?delivery?problems?(Klesius?and?Shoemaker?1998).?In?addition,?there?is?the?potential?for?antibiotic?resistance?to?develop.??Plasmid??
mediated?resistance?of?several?strains?ofE.?ictalurito?Romet?has?been?reported?(Starliper?et?al.1993).?At?one?time,?the?efectiveness?of?Romet?was?reduced?due?to?palatability?
problems,?but?that?problem?has?since?been?corected.?Poe?and?Wilson?(1989)?found?that
30?
feed?consumption?decreased?as?the?amount?of?Romet?incorporated?into?feed?increased,?due?to?palatability?problems?caused?by?ormetroprim.?A?study?by?Robinson?et?al.?(1990)?
showed?that?the?problem?could?be?corected?by?increasing?the?amount?of?fish?meal?in?the?feed?from?4%?to?16%.?
Florfenicol?has?been?shown?to?be?efective?in?treating?ESC?and?palatable?when?incorporated?into?feed?in?two?separate?studies?conducted?by?Gaunt?et?al.?(2003).?In?the?
palatability?study,?the?researchers?placed?channel?catfish?into?tanks,?and?over?a?10?d?period,?fed?them?feed?containing?diferent?amounts?of?florfenicol,?such?that?fish?were?
ofered?0,?10,?20,?40,?or?100?mg?of?florfenicol?per?kg?of?body?weight.?Each?day?tanks?were?given?a?numerical?score?based?upon?feeding?activity,?and?at?the?end?of?the?10?d?
feeding?period,?these?scores?were?used?to?compute?palatability?scores?for?each?treatment?group.?At?the?end?of?the?10?d?feeding?period,?fish?were?removed?from?the?tanks?and?
weighed.?No?significant?diferences?were?observed?in?palatability?scores?or?weight?gain?among?the?treatment?groups.?In?the?eficacy?study,?Gaunt?et?al.?(2003)?stocked?channel?
catfish?into?tanks?and?compared?mortalities?among?five?treatment?groups:?(1)?not?challenged?withE.?ictaluri?and?fed?unmedicated?feed,?(2)?chalenged?withE.?ictaluri?and?
fed?unmedicated?feed,?(3)?chalenged?and?fed?florfenicol?at?the?rate?of?10?mg?per?kg?of?body?weight,?(4)?chalenged?and?fed?florfenicol?at?the?rate?of?20?mg?per?kg?of?body?
weight,?and?(5)?chalenged?and?fed?florfenicol?at?the?rate?of?40?mg?per?kg?of?body?weight.?The?cumulative?mortality?rate?for?each?treatment?group?was?2.5,?57.5,?0,?1.25,?and?1.25%,?
respectively,?such?that?the?mortality?rate?of?each?treated?group?was?significantly?lower?than?the?untreated,?chalenged?group.
31?
Fish?farmers?have?attempted?to?control?ESC?by?restricting?the?amount?offeed?ofered.?Wise?and?Johnson?(1998)?confirmed?the?efectiveness?of?practicing?restricted?
feeding?in?controlling?ESC.?In?their?study,?fish?were?completely?withheld?from?feed??ofered?medicated?feed?for?five?consecutive?days?and?non?medicated?feed?thereafter??or?
ofered?medicated?or?non?medicated?feed?every?day,?every?other?day,?or?every?third?day.?Fish?completely?withheld?from?feed?or?ofered?Romet?medicated?feed?every?other?day?or?
every?third?day?had?the?highest?survival.?Wise?and?Johnson?(1998)?hypothesized?that?a?reduction?in?the?amount?of?feed?ofered?limits?exposure?to?E.?ictalurithrough?ingestion.?
Although?restricted?feeding?can?mitigate?the?efects?of?ESC,?it?results?in?a?los?of?production?(Wise?et?al.?2000,?2004).?
Fish?farmers?have?also?attempted?to?control?ESC?by?practicing?good?management?(Tucker?and?Robinson?1990).?Lack?of?adequate?nutrients?and?stress?can?result?in?
immunosuppression.?Therefore,?providing?fod?with?adequate?nutrients,?acclimating?fish?to?water?properly,?and?maintaining?good?water?quality?reduce?incidences?and?severity?of?
ESC.?Due?to?the?lack?of?eficient?control?measures,?interest?in?developing?a?vaccine?for?
ESC?began?growing?in?the?late?1980s.?Vaccines?had?been?in?use?in?the?salmonid?and?trout?industry?for?years,?beginning?in?the?early?1960s?with?the?development?of?the?first?
practical,?comercial?vaccine?for?enteric?redmouth?(Ros?and?Klontz?1965).?However,?a?serious?obstacle?to?developing?a?vaccine?was?the?heterogeneous?serology?of?most?
pathogens?afecting?fish?(Thune?et?al.?1994).?Fortunately,?early?studies?indicated?that?isolates?ofE.?ictaluri?were?biochemically?and?serologically?homogenous.?Biochemical?
characterization?of?119?isolates?ofE.?ictaluri?revealed?very?few?diferences?among?them
32?
(Waltman?et?al.?1985).?Plumb?and?Vinitnantharat?(1989)?examined?40?isolates?and?found?very?few?biochemical?or?serological?diferences.?A?later?study?by?Panangala?et?al.?(2005)?
showed?E.?ictaluriisolates?to?be?genetically?homogenous,?with?respect?to?the?16S?23S?rRNA?intergenic?spacer?region?(ISR).?The?researchers?amplified?the?16S?23S?rRNA?ISR?
sequences?of?19?E.?ictaluri?isolates?by?polymerase?chain?reaction,?and?after?performing?sequence?analyses,?found?no?diferences?among?the?ISR?sequences.?
Another?chalenge?facing?researchers?was?that?it?would?be?necessary?to?administer?any?vaccine?developed?for?channel?catfish?orally?or?by?immersion.?Vaccination?in?
salmonid?and?trout?culture?had?typically?been?performed?by?intraperitoneal?injection?of?bacterins?(Klesius?and?Shoemaker?1998).?Intraperitoneal?injection?generates?large?
immune?responses,?because?it?alows?for?the?use?of?adjuvants?(Elis?1988)?and?guarantees?that?each?fish?in?a?group?is?exposed?to?and?receives?approximately?the?same?number?of?
bacteria?(Ciembor?et?al.?1995).?The?high?individual?worth?of?salmonids?and?trout?had?alowed?for?high?labor?costs?associated?with?injecting?each?fish.?On?the?other?hand,?the?
low?individual?worth?of?channel?catfish?would?prohibit?development?of?vaccines?administered?by?injection.?
Development?of?a?Vaccine?Vaccines?are?preparations?of?nonpathogenic?microbial?antigen,?either?dead?or?live,?
that?are?administered?by?injection,?orally,?or?by?immersion.?Dead?antigens?may?consist?of?microbial?extract,?inactivated?organisms,?or?bacterins?that?are?prepared?by?heat?or?
formalin?treatment.?Live?bacteria?must?be?attenuated?to?render?them?nonpathogenic.?Vaccines?protect?against?disease?by?inducing?the?adaptive?immune?system?to?develop?
long?lived?efector?and?memory?cells.?Although?vaccines?protect?against?disease,?they?do
33?
not?necessarily?protect?against?establishment?of?an?asymptomatic?carier?state?(Ellis?1988).?
Oral?and?Imersion?Bacterin?Vaccine?Trials?Folowing?the?succes?of?bacterin?vaccines?developed?for?coldwater?species,?
immersion?and?oral?bacterins?were?developed?for?vaccinating?channel?catfish?against?ESC.?An?immersion?bacterin?was?developed?by?BIOMED?Inc.?(Belevue,?WA,?USA)?
(Shoemaker?and?Klesius?1997).?An?oral?bacterin?was?developed?by?Escogen,?Aqua?Health?Ltd.?(Charlestown,?PEI,Canada)?(Shoemaker?and?Klesius?1997).?
Oral?and?immersion?bacterins?failed?to?consistently?provide?protection.?An?early?study?by?Plumb?and?Vinitnantharat?(1993)?found?bacterin?vaccines?to?provide?protection.?
The?researchers?stocked?channel?catfish?fryvaccinated?by?immersion?or?imersion?folowed?by?an?oral?boster?into?ponds.?Some?fish?were?removed?before?harvest?and?
subjected?to?a?laboratory?chalenge.??The?relative?percent?survival?(RPS)?in?fish?vaccinated?by?immersion?or?immersion?folowed?by?an?oral?boster?was?93.1?and?96.6,?
respectively.??At?harvest?survival?of?groups?vaccinated?by?immersion?folowed?by?an?oral?boster?was?higher?than?in?groups?vaccinated?by?immersion?alone?and?in?control?groups.?
Thune?et?al.?(1994)?had?mixed?succeses?with?bacterin?vaccine?trials.?In?a?preliminary?pond?study,?the?authors?vaccinated?channel?catfish?fry?by?immersion?or?
orally,?stocked?them?into?ponds,?and?gave?an?oral?booster?to?a?portion?of?the?fry?vaccinated?by?immersion.?Some?fish?were?removed?before?harvest?and?subjected?to?a?
laboratory?challenge.??Only?vaccination?by?immersion?resulted?in?an?RPS?value?above?50.?At?harvest?no?significant?diference?in?survival?was?observed?among?treatment?groups.
34?
In?a?pond?study?conducted?in?1987?1988,?Thune?et?al.?(1994)?vaccinated?channel?catfish?fry?by?immersion?or?orally,?stocked?them?into?ponds,?and?ofered?an?oral?boster?
to?a?portion?of?the?fry?vaccinated?by?immersion.?At?harvest,?RPS?values?were?calculated?to?be?57.4,?50.3,?and?53.5,?respectively,?for?fish?vaccinated?by?immersion,?orally?or?by?
immersion?folowed?by?an?oral?boster.?In?two?separate?pond?studies?conducted?in?1989??1990?and?1990?1991,?Thune?et?al.?(1994)?vaccinated?fry?by?immersion,?stocked?them?into?
ponds,?and?ofered?them?an?oral?boster.?In?the?pond?study?conducted?in?1989?1990,?no?significant?diference?in?survival?was?observed?between?vaccinated?and?non?vaccinated?
fish.?In?the?pond?study?in?1990?1991,?survival?was?significantly?higher?in?vaccinated?fish,?but?the?overal?RPS?value?was?only?35.1.?
In?later?unpublished?laboratory?studies,?Thune?et?al.?was?unable?to?reproduce?any?of?the?successes?in?their?earlier?pond?studies?(Thune?et?al.?1997).?Thune?et?al.?(1994)?
suggested?the?apparent?success?of?the?vaccine?in?the?1987?1988?field?trial?might?have?been?due?to?the?ability?of?naturally?occurring?E.?ictaluri?to?stimulate?primary?or?secondary?
immune?responses.?This?suggestion?may?also?serve?to?explain?the?succeses?observed?in?the?field?study?of?Plumb?and?Vinitnantharat?(1993),?who?isolated?E.?ictaluri?from?two?
apparently?healthy?fish?from?their?study?indicating?that?E.?ictaluri?was?present?in?ponds?used?for?their?study.?
Other?researchers?also?had?mixed?succes?with?bacterin?vaccine?trials.?Plumb?et?al.?(1994)?stocked?fry?into?earthen?ponds?at?low,?intermediate,?or?high?densities,?and?later?
vaccinated?them?orally.?At?harvest,?survival?was?not?significantly?higher?in?vaccinated?fish?at?any?of?the?stocking?densities.?Some?fish?from?harvest?were?subjected?to?a
35?
laboratory?challenge.??An?RPS?value?greater?than?50?was?observed?only?in?vaccinated?fish?stocked?at?the?highest?density.?
Shoemaker?and?Klesius?(1997)?stocked?juvenile?channel?catfish?into?aquaria,?vaccinated?them?by?immersion?or?oral?bacterins,?and?later?chalenged?them.?No?
significant?diference?in?survival?was?observed?between?fish?vaccinated?by?either?regime?and?non?vaccinated?fish.?In?a?second?experiment,?Shoemaker?and?Klesius?(1997)?
challenged?fish?vaccinated?by?immersion?bacterin.?No?significant?diference?in?survival?was?observed?between?vaccinated?fish?and?non?vacinated?fish.?In?a?third?experiment?
Shoemaker?and?Klesius?(1997)?chalenged?fish?vaccinated?by?immersion?bacterin?folowed?by?an?oral?bacterin?boster.?Again,?no?significant?diference?in?survival?was?
observed?between?vaccinated?fish?and?non?vaccinated?fish.?Inconsistent?success?with?immersion?and?oral?bacterin?trials?may?have?been?
observed?for?a?variety?of?reasons.?The?heat?inactivation?needed?to?prepare?both?immersion?and?oral?bacterins?often?severely?denatures?surface?proteins?(Nusbaum?and?
Morison?1996).?Oral?bacterins?tend?to?expose?fish?to?diferent?doses?of?antigen,?due?to?fish?consuming?diferent?amounts?of?feed?(Elis?1988)??are?often?destroyed?within?the?
digestive?system?before?the?immune?response?has?time?to?respond?(Elis?1988)??and?may?leach?from?feed?into?surrounding?water?(Thune?et?al.?1994).?Even?if?antigen?does?make?it?
through?the?stomach?without?being?destroyed,?it?may?not?be?absorbed?in?the?intestines.?Shoemaker?and?Klesius?(1997)?suggested?that?immersion?bacterins?may?fail?to?expose?
fish?to?a?sufficient?amount?of?antigen?and?macrophages?may?be?unable?to?proces?bacterin?and?present?it?to?B?cels.?Results?from?a?study?conducted?by?Nusbaum?and?Morison?
(1996)?supported?the?former?suggestion?of?Shoemaker?and?Klesius?(1997).?Nusbaum?and
36?
Morison?(1996)?immersed?channel?catfish?in?water?containing?heat?killed?radiolabeled?E.?ictaluri?and?attempted?to?detect?the?presence?of?radiolabeled?bacteria?in?various?organs?
where?E.?ictaluritypically?enters?(i.e.?nares,?gils,?and?gut).?No?levels?of?radiolabeled?bacteria?above?background?levels?could?be?detected,?leading?the?authors?to?suggest?the?
need?to?reasess?the?efectivenes?of?oral?and?immersion?bacterins.?Probably?the?most?significant?factor?contributing?to?the?inconsistent?success?of?oral?and?immersion?bacterins?
was?their?inability?to?induce?a?cel?mediated?imune?response?(Klesius?1992a),?which?plays?a?major?role?in?protecting?against?intracelular?pathogens?(Abbas?and?Lichtman?
2003e).? Virulent,?Live?Vaccine?Trials?
Due?to?the?failure?of?bacterins?to?provide?consistent?protection,?the?search?for?an?efective?live?vaccine?was?begun.?Unlike?bacterins,?live?vaccines?are?capable?of?inducing?
both?the?humoral?and?cell?mediated?components?of?immunity?(Klesius?1992a).?Vaccination?of?channel?catfish?by?immersion?in?virulent,?live?E.?ictaluri?was?found?to?
result?in?protection?against?later?exposures.?Shoemaker?and?Klesius?(1997)?stocked?juvenile?channel?catfish?into?aquaria,?vaccinated?them?with?virulent,?live?E.?ictaluri,?and?
later?chalenged?them.?Survival?in?vaccinated?fish?was?significantly?higher?than?in?non??vaccinated?fish.?Klesius?and?Shoemaker?(1997)?immunized?channel?catfish?with?a?single?
isolate?of?virulent,?live?E.?ictaluri?and?later?chalenged?them?with?the?same?isolate.??An?RPS?value?of?88.3?was?calculated.?
Although?virulent,?live?vaccines?were?found?to?be?protective?against?ESC,?isolates?were?shown?to?vary?in?their?ability?to?provide?protection.?Klesius?and?Shoemaker?(1997)?
immunized?channel?catfish?with?a?single?isolate?of?virulent,?live?E.?ictaluri?and?later
37?
challenged?them?with?heterologous?isolates.?Protection?was?not?observed?against?challenge?with?one?of?the?isolates.?In?a?separate?experiment,?channel?catfish?were?
immunized?with?heterologous?isolates?of?virulent,?live?E.?ictaluri,?and?later?chalenged?with?a?single?isolate.??Not?al?isolates?used?for?immunization?resulted?in?protection?against?
the?single?isolate.??The?results?of?both?experiments?led?the?authors?to?suggest?that?oral?vaccination?trials?of?the?past?(Thune?et?al.?1994??Thune?et?al.?1997??Plumb?et?al.?1994??
Shoemaker?and?Klesius?1997)?may?have?failed?due?to?oral?bacterins?consisting?of?only?one?isolate.?
The?results?of?Klesius?and?Shoemaker?(1997)?seemed?to?be?inconsistent?with?studies?indicating?that?isolates?ofE.?ictaluri?were?homogenous?isolates?(Waltman?et?al.?
1986??Plumb?and?Vinitnantharat?1989).?However,?Klesius?and?Shoemaker?(1997)?suggested?that,?although?isolates?may?be?homogenous?according?to?established?techniques?
designed?for?assessing?the?homogeneity?of?mammalian?pathogens,?fish?may?not?necessarily?recognize?them?as?being?homogenous?(Mutheria?et?al.?1993??Hasting?and?Ellis?
1988).? Attenuated,?Live?Vaccine?Trials?
Although?exposure?to?virulent?E.?ictaluri?was?shown?to?induce?a?cell?mediated?response?and?provide?protection?against?subsequent?exposures,?using?virulent,?live?E.?
ictaluri?as?a?vaccine?obviously?would?have?failed?to?fulfill?the??safe??criterion?of?an?efective?vaccine.??Therefore,?the?search?for?an?attenuated,?live?vaccine?that?would?not?
result?in?significant?mortalities?upon?administration?was?begun.?In?time?Klesius?and?Shoemaker?(1998)?produced?a?live,?attenuated?E.?ictaluri?vaccine?(E.?ictaluri?RE?33).
38?
Intervet,?Inc.???licensed?and?produced?the?vaccine?under?the?name?AQUAVAC?ESC???(Shoemaker?et?al.?2002).?
In?considering?how?to?attenuateE.?ictaluri,?Klesius?and?Shoemaker?(1998)?took?into?account?that?other?researchers?were?able?to?produce?an?efective?vaccine?against?
furunculosis?in?rainbow?trout?by?removing?the?O?side?chain?of?LPS?inAeromonas?salmonicida?(Thornton?et?al.?1994).??The?researchers?also?took?into?account?the?findings?
of?Klesius?and?Shoemaker?(1997),?which?indicated?that?a?successful?vaccine?would?be?composed?of?heterologous?isolates.?Therefore,?Klesius?and?Shoemaker?(1998)?
hypothesized?that?an?O?antigen?deficient?E.?ictaluri?vaccine?would?protect?channel?catfish?against?ESC.?To?remove?the?O?side?chain,?a?weakly?virulent?isolate?ofE.?ictaluri(EILO)?
originally?isolated?from?walking?catfish?in?Thailand,?was?passed?through?increasing?concentrations?of?rifampicin?33?times.?The?O?antigen?deficient?EILO?isolate?was?
designated?RE?33.?Klesius?and?Shoemaker?(1998)?demonstrated?the?safety?of?the?RE?33?vaccine.?
Channel?catfish?(3?9?mos?of?age)?were?vaccinated?by?immersion?at?200?times?the?vaccine?dose?(2.0?x?10?7?CFU/mL)?for?30?times?longer?than?the?vaccine?exposure?time?(60?min).?
No?mortalities?or?clinical?signs?arose?folowing?immersion.?Procedures?were?performed?to?determine?if?reversion?to?virulence?occurred.?No?reversion?was?observed.?
Atenuated,?live?vaccines?induce?low?level?infections?that?persist?in?the?host?for?a?somewhat?lengthy?period?of?time,?and?then?are?cleared?by?the?host.?Persistence?in?the?
host?provides?continual?antigenic?stimulation,?and?therefore,?greater?imunity?than?if?the?vaccine?did?not?persist?for?longer?periods?of?time.??Clearance?is?obviously?necesary?for?
safety?purposes?(Wise?and?Terhune?2001).?Therefore,?Klesius?and?Shoemaker?(1998)
39?
evaluated?survival?and?clearance?ofE.?ictaluri?after?administration?of?the?vaccine.??Their?evaluation?revealed?that?E.?ictaluri?survived?in?vacinates?only?14?d?before?being?cleared.?
They?attributed?early?clearance?to?their?unpublished?findings?that?complement?was?more?efective?in?killing?E.?ictaluri?RE?33?than?virulent?E.?ictaluri.?
The?ability?of?the?RE?33?vaccine?to?induce?long?lasting?protection?under?laboratory?conditions?was?shown?(Klesius?and?Shoemaker?1998).?Channel?catfish?3?9?
mos?of?age?were?vaccinated?by?immersion?in?RE?33?at?1.0?x?10?5?CFU/mL?for?2?min?and?challenged?with?13?isolates?of?virulent?E.?ictaluri?14?d?post?vaccination.?The?RE?33?
vaccine?was?succesful?in?providing?protection?to?8?of?13?isolates.?When?challenge?occurred?at?a?later?time,?protection?was?demonstrated?intwo?additional?isolates.?
Protection?lasted?at?least?4?mos,?as?demonstrated?by?challenge.?A?significant?point?is?that?E.?ictaluri?RE?33?was?not?found?to?induce?large?antibody?responses,?which?supports?
earlier?studies?showing?a?lack?of?or?negative?corelation?between?antibody?titers?and?survival.?
The?AQUAVAC?ESC?vaccine?has?also?been?shown?to?provide?protection?under?laboratory?conditions?when?administered?to?young?fry.?The?ability?to?vaccinate?fry?before?
moving?them?into?production?ponds?is?desirable,?because?it?is?dificult?to?recover?fry?once?they?have?been?released?(Petrie?Hanson?and?Ainsworth?1999).?Wise?and?Terhune?(2001)?
vaccinated?channel?catfish?fry?at?12?d?post?hatch?and?later?chalenged?them.?Survival?was?significantly?higher?in?vaccinated?fry?than?in?control?fry.??Shoemaker?et?al.?(1999)?
vaccinated?channel?catfish?fry?by?immersion?at?7?d?post?hatch?and?later?challenged?them.?Relative?percent?survival?ranged?from?58.4?to?77.5.
40?
The?AQUAVAC?ESC?has?even?been?shown?to?provide?protection?when?administered?in?ovo.?Shoemaker?et?al.?(2002)?vaccinated?eyed?channel?catfish?eggs?and?
challenged?hatch?fry?60?d?post?vaccination.?Relative?percent?survival?was?59.7%.?Prior?to?studies?indicating?that?E.?ictaluri?RE?33?could?induce?protection?in?young?
fry,?questions?had?arisen?as?to?whether?the?imune?system?of?young?fry?was?developed?enough?to?respond?to?a?vaccine.??Petrie?Hanson?and?Ainsworth?(1999)?vaccinated?channel?
catfish?fry?at?1,?2,?3?or?4?weeks?of?age?or?2,?3,?4,?5,?or?6?mos?of?age?with?live?E.?ictaluri?and?assayed?antibodies?2?weeks?post?vaccination.?A?humoral?immune?response?greater?
than?that?of?controls?was?not?observed?in?fish?younger?than?4?weeks?of?age.?However,?studies?have?shown?that?cel?mediated?immunity,?not?humoral?immunity,?is?important?in?
providing?protection?against?ESC.?Wise?and?Terhune?(2001)?suggested?that?E.?ictaluri?RE?33?may?persist?in?tisues?until?the?immune?system?fully?develops.?As?support,?they?
pointed?out?the?positive?corelation?between?exposure?dose,?which?relatesto?the?establishment?of?low?level?infections,?and?survival?to?challenge?with?a?virulent?strain?in?
their?study.?The?succes?of?the?AQUAVAC?ESC?vaccine?has?been?shown?to?depend?on?
exposure?dose.??Wise?et?al.?(2000)?vaccinated?channel?catfish?fingerlings?72?d?post?hatch?by?a?30?min?bath?immersion?at?concentrations?of?1.0?x?10?6?,?1.0?x?10?7?,?or?2.0?x?10?7?
CFU/mL?and?later?chalenged?them?in?the?laboratory?and?in?the?field?in?floating?net?pens.?Survival?was?significantly?higher?in?fish?vaccinated?at?the?highand?intermediate?doses?as?
compared?to?the?low?dose?in?both?the?laboratory?and?field?challenges.?No?significant?diference?was?observed?in?survival?between?fish?vaccinated?at?the?intermediate?and?high?
doses.?Wise?and?Terhune?(2001)?vaccinated?channel?catfish?fry?at?12?d?post?hatch?by?a
41?
2?min?bath?immersion?at?doses?of?2.5?x?10?5?,?2.5?x?10?6?,?or?2.4?x?10?7?CFU/mL?and?later?challenged?them.?The?researchers?were?able?to?recover?E.?ictaluri?RE?33?from?a?greater?
percentage?of?fry?vaccinated?at?the?high?and?intermediate?doses?than?fry?vaccinated?at?the?low?dose.?Regardles?of?dose,?the?researchers?were?able?to?recover?the?bacterium?from?
the?greatest?percentage?of?fry?between?1?and?6?d?post?vaccination.?After?challenge,?survival?was?significantly?higher?in?fry?vaccinated?at?the?high?and?intermediate?doses?than?
in?fry?vaccinated?at?the?low?dose?and?control?fish.?No?significant?diference?in?survival?was?observed?between?fry?vaccinated?at?the?low?dose?and?control?fish.?
Evaluation?of?the?AQUAVAC?ESC?vaccine?under?pond?conditions?has?shown?mixed?results.?Wise?et?al.?(2000)?vaccinated?channel?catfish?fingerlings?72?d?post?hatch?
at?1.0?x?10?7?CFU/mL,?and?later?challenged?them?in?the?laboratory?and?in?the?field?in?floating?net?pens.?In?both?the?field?and?laboratory?challenges,?survival?was?significantly?
higher?in?vaccinated?fish?than?that?in?control?fish.??However,?data?from?a?study?by?Carrias?(2005),?in?which?the?efect?of?vaccinating?fry?with?AQUAVAC?ESC?on?survival?was?
evaluated?under?normal?pond?conditions,?was?inconclusive.?Carrias?(2005)?vaccinated?channel?catfish?fry?at?10?d?post?hatch?or?32?d?post?hatch,?stocked?them?into?primary?
nursery?tanks,?and?grew?them?out?to?fingerling?stage.?Mean?survival?of?32?d?vaccinates?was?significantly?higher?than?controls,?but?mean?survival?of?10?d?vaccinates?was?not?
significantly?higher.?The?author?suggested?that?a?significant?diference?in?survival?of?the?10?d?vaccinates?may?not?have?been?observed,?due?to?columnaris?infections?mixed?with?
ESC.?For?a?fish?vaccine?to?be?economically?beneficial,?savings?associated?with?the?
vaccine?must?outweigh?costs?of?administering?the?vaccine.?Lilehaug?(1989)?developed?a
42?
formula?to?calculate?the?break?even?point?between?costs?of?vaccination?and?economic?loses?due?to?disease.??Factors?used?to?calculate?costs?associated?with?administering?the?
vaccine?include:?the?man?hours?required?for?the?vaccination?method,?workers??wages,?the?volume?of?vaccine?consumed,?the?price?of?the?vaccine?per?liter,?and?additional?costs?
(necesary?equipment,?anesthetics,?and?the?value?of?fish?lost?due?to?side?efects).?Factors?used?to?calculate?savings?associated?with?vaccine?include:?the?expected?mortality,?the?
RPS?of?the?vaccine,?the?mean?weight?of?fish?at?slaughter,?the?price?obtained?for?the?fish?per?kg,?the?FCR,?and?the?price?of?feed?per?kg.?The?formula?does?not?include?the?posible?
savings?in?reduced?drug?costs?associated?with?treating?an?outbreak?of?the?disease.?To?date?no?studies?have?evaluated?under?pond?conditions?the?relative?survival?of?
channel?catfish?immunized?with?AQUAVAC?ESC?as?fingerlings.?Administration?of?the?vaccine?to?fingerlings?at?the?dose?recomended?by?Intervet?Inc.?currently?is?not?
economically?feasible,?because?the?weight?of?the?fish?and?the?volume?of?water?required?to?hold?the?fish?requires?a?considerable?volume?of?vaccine.?However,?fingerlings?can?be?
immersed?in?vaccine?for?longer?periods?of?time?than?fry,?because?fry?are?more?susceptible?to?crowding?stress?than?fingerlings?(Tucker?and?Robinson?1990).?Therefore,?increasing?
vaccine?immersion?time?from?the?17?min?period?currently?recomended?to?a?4?or?8?h?period?may?allow?a?lower?dose?of?vaccine?to?provide?protection?against?ESC.?
Administering?the?vaccine?to?fingerlings?would?be?beneficial?in?that?it?would?alow?farmers?the?option?of(1)?vaccinating?na?ve?fingerlings?while?moving?them?to?food?fish?
ponds?and?(2)?giving?a?boster?of?the?vaccine?to?fingerlings?previously?vaccinated?as?fry.?Low?costs?would?be?associated?with?administering?the?vaccine?to?fingerlings.?
Fingerlings?are?often?transported?from?one?farm?to?another?for?periods?of?approximately
43?
4?h,?leaving?open?the?posibility?of?administering?the?vaccine?in?route.?In?route?vaccination?would?require?no?additional?man?hours,?no?anesthetics,?and?no?additional?
equipment.?Vaccination?of?fingerlings?can?easily?be?integrated?into?the?production?cycle,?as?they?are?already?being?handled?and?gathered?up?for?transfer?from?fingerling?ponds?to?
fod?fish?ponds.?Administering?the?vaccine?to?fingerlings?may?provide?savings?in?that,?although?fod?fish?typically?sufer?low?mortalities?from?the?chronic?form?of?ESC,?larger?
fish?are?worth?more?per?individual?than?smaller?fish?(Wise?et?al.?2004).?The?objectives?of?this?study?were?to?(1)?evaluate?a?more?economically?feasible?dose?of?AQUAVAC?ESC?to?
administer?to?channel?catfish?fingerlings?for?protection?against?ESC,?(2)?evaluate?under?normal?pond?conditions?the?relative?survival?of?fish?immunized?as?fry?with?AQUAVAC??
ESC?and?given?a?boster?of?the?vaccine?as?fingerlings,?and?(3)?evaluate?the?long?term?benefits?of?vaccination?to?determine?if?a?boster?is?necessary?for?life?long?protection.
44?
MATERIALS?AND?METHODS?
Laboratory?Experiment?1?Channel?catfish?fingerlings?(NWAC?103?strain,?~?34?g)?were?stocked?into?18,?
32?L?aquaria?at?a?density?of?ten?fish/tank?and?water?temperature?maintained?at?24?C.?Fish?used?for?the?experiments?had?been?maintained?in?flow?through?tanks,?with?water?derived?
from?a?wel,?at?the?North?Auburn?S6?laboratory.?No?previous?outbreaks?had?occurred?among?the?fish,?nor?had?any?samples?tested?positive?for?E.?ictaluri?specific?antibodies.?
Fish?were?fed?to?satiation?daily?throughout?the?duration?of?the?experiment.?Water?flow?to?each?tank?was?shut?of?1?h?prior?to?vaccination,?and?salt?was?added?to?reach?a?chloride?
concentration?of?~?2?ppt.?According?to?manufacturer?s?directions,?one?vial?of?vaccine?reconstituted?in?50?mL?of?water?and?added?to?3.78?L?of?water?creates?an?approximate?
concentration?of?1.0?x?10?7?CFU/mL.?Fish?were?vaccinated?at?target?concentrations?of?1.0?x?10?4?,?1.0?x?10?5?,?or?1.0?x?10?6?CFU/mL?by?adding?0.43,?4.3,?or?43?mL?of?reconstituted?
vaccine,?respectively,?to?tanks.?Water?flow?to?each?tank?was?resumed?either?4?or?8?h?post?vaccination.?Six?treatment?groups?(three?replicate?tanks?each)?were?created?ina?3x2?
factorial?design,?with?the?main?factors?being?dose?and?time.?Reconstituted?vaccine?was?enumerated?by?standard?plate?count?methods?on?brain?heart?infusion?(BHI)?agar?incubated?
at?27?C?for?48?h.?Water?samples?(1.0?mL)?from?each?replicate?aquaria?within?each
45?
treatment?were?pooled?and?vaccine?isolate?enumerated?as?above.?Final?vaccine?dose?concentrations?were?2.44?x?10?4?,?3.48?x?10?5?,?or?3.16?x?10?6?CFU/mL.?
Nineteen?days?post?vaccination?serum?was?colected?from?five?fish?per?tank.?Fish?were?anaesthetized?with?Finquel?(Argent?Chemical?Laboratories,?Redmond,?Washington,?
USA)?and?blod?was?colected?from?the?caudal?sinuses?using?Vaccutainer???tubes?(Becton?Dickinson?and?Co.,?Franklin?Lakes,?New?Jersey,?USA).?Blod?samples?were?held?at?
room?temperature?for?1?h?and?thereafter?refrigerated?for?24?h.?Serum?from?samples?was?decanted?into?microfuge?tubes?and?centrifuged?at?1000?rpm?for?5?min?to?remove?any?
remaining?celular?debris.?Serum?from?centrifuged?tubes?was?decanted?into?a?second?set?of?microfuge?tubes?and?frozen?at??80?C?for?later?analysis.?
Antibody?titers?of?colected?serum?samples?were?determined?by?an?agglutination?assay?described?by?Contrath?(1972).?A?two?fold?dilution?series,?using?PBS?with?0.05%?
tween?80,?was?prepared?for?each?sample?in?a?96?wel?plate.?Two?positive?and?two?negative?control?wels,?using?known?positive?and?negative?sera,?were?included.?A?
virulent?strain?(S97?773)?ofE.?ictaluri?was?grown?to?stationary?phase?and?killed?with?2%?formalin?to?serve?as?antigen.?Equal?amounts?of?antigen?were?added?to?each?well?in?the?
dilution?series.?Plates?were?alowed?to?sit?at?room?temperature?overnight.?The?reciprocal?of?the?highest?serum?dilution?at?which?agglutination?occurred?was?considered?to?be?the?
antibody?titer?of?the?sample.? Laboratory?Experiment?2?
Channel?catfish?fingerlings?(NWAC?103?strain,?~34?g)?were?stocked?into?nine,?32?L?aquaria?at?a?density?of?15?fish/tank.?Fish?were?acclimated?at?18?C?for?two?weeks?
and?maintained?at?this?temperature?for?the?duration?of?the?experiment.?Fishwere?fed?to
46?
satiation?daily.?Water?flow?to?each?tank?was?shut?off?1?h?prior?to?vaccination,?and?salt?was?added?to?reach?a?chloride?concentration?of?~?2?ppt.?Fish?were?vaccinated?at?target?
concentrations?of?1.0?x?10?4?,?1.0?x?10?5?,?or?1.0?x?10?6?CFU/mL?according?to?the?procedures?described?previously.?Water?flow?to?each?tank?was?resumed?4?h?post?vaccination.?
Enumeration?procedures?for?the?vaccine?were?folowed?as?previously?described.?Reconstituted?vaccine?concentration?was?determined?to?be?1.5?x?10?9?CFU/mL.?Twenty??
one?days?post?vaccination?serum?was?colected?from?ten?fish?per?tank?and?agglutinating??antibody?titers?were?determined?according?to?the?procedures?described?above.?
Laboratory?Experiment?3?Channel?catfish?fingerlings?(NWAC?103?strain,?~?15?g)?were?stocked?into?20,?
32?L?tanks?at?a?density?of?12?fish/tank.?Fish?were?fed?to?satiation?daily?throughout?the?duration?of?the?experiment.?Water?level?in?each?tank?was?reduced?to?15?L,?flow?was?shut?
of?1?h?prior?to?vaccination,?and?salt?was?added?to?reacha?chloride?concentration?of?~?2?ppt.?According?to?manufacturer?s?directions,?one?vial?of?frozen?vaccine?added?to?19?L?of?
water?creates?an?approximate?concentration?of?1.0?x?10?7?CFU/mL.?A?stock?of?vaccine?was?made?by?adding?three?vials?to?5.7?L?of?waterto?create?an?approximate?concentration?
of?1.0?x?10?8?CFU/mL.?Four?treatment?groups?(five?replicate?aquaria?each)?were?created?by?vaccinating?fish?at?target?concentrations?of?1.0?x?10?4?,?1.0?x?10?5?,?or?1.0?x?10?6?CFU/mL?
by?adding?0.15,?15,?or?150?mL?of?vaccine?stock,?respectively,?to?replicate?tanks.?Control?tanks?were?also?created?in?which?no?vaccine?was?added.?Water?flow?to?each?tank?was?
resumed?after?4?h?post?vaccination.?Water?temperature?at?time?of?vaccination?was?25?C.?Final?vaccine?concentrations?in?tanks?were?determined?to?be?1.95?x?10?4?,?3.80?x?10?5?,?or?
1.95?x?10?6?CFU/mL.
47?
Sixty?days?post?vaccination,?fish?were?challenged?with?a?virulent?strain?ofE.?ictaluri?(S97?775)?for?1?h.?A?culture?ofE.?ictaluriwas?grown?in?BHI?broth?at?25?C?for?
24?h.?Subsequent?plating?of?serial?dilutions?of?the?challenge?culture?revealed?that?fish?were?challenged?at?a?concentration?of?1.2?x?10?5?CFU/mL.?Dead?fish?were?removed?daily,?
and?necropsies?were?performed?on?at?least?five?suitable?mortalities?from?each?tank?to?verifyE.?ictaluri?infection.?Bacterial?pathogens?were?isolated?from?the?liver?on?BHI?and?
Hsu?Shotts?agar?plates?to?test?for?the?presence?ofE.?ictalurior?Flavobacterium?columnare.?Negative?results?for?indole?production?and?cytochrome?oxidase?biochemical?
tests?were?used?to?confirm?the?presence?ofE.?ictaluri?(Hawke?et?al.?1981).?The?challenge?was?stopped?after?16?d.?
Pond?Study?Twenty?0.04?ha?ponds?at?the?Auburn?University?Fisheries?Experiment?Station?
were?randomly?assigned?to?one?of?the?four?treatments.?One?week?prior?to?stocking,?powdered?agricultural?limestone?was?added?to?each?pond?at?a?rate?of?2,260?kg/ha,?and?
then?ponds?were?filled?with?water?from?a?reservoir,?with?screens?placed?on?inlet?pipes?to?prevent?entry?of?wild?fish.?
Channel?catfish?fingerlings?(NWAC?103?strain)?used?in?a?previous?study?(Carrias?2005)?were?harvested?from?ponds?located?at?the?North?Auburn?Fisheries?Research?Station?
for?use?in?the?present?study.?Fingerlings?had?been?divided?among?individual?ponds,?according?to?two?treatment?groups,?and?over?wintered?since?December?2004.?Four?ponds?
contained?fingerlings?vaccinated?with?AQUAVAC?ESC???as?fry?at?10?d?of?age?and?four?ponds?contained?fingerlings?non?vaccinated?as?fry.?Fingerlings?from?al?four?ponds?in?
each?treatment?were?pooled?together?in?holding?tanks?and?passed?through?a?series?of
48?
bar?graders?to?sort?the?fish?according?to?size.?Retained?fingerlings?(~?39?g)?were?transferred?from?the?holding?tanks?to?experimental?ponds?via?hauling?tanks.?Within?
hauling?tanks,?vaccine?was?administered?to?half?of?the?fingerlings?that?were?non??vaccinated?as?fry?in?the?previous?study?and?half?of?the?fingerlings?vaccinated?as?fry?at?10?
d?post?hatch.?Fingerling?fish?were?vaccinated?for?4?h?in?250?L?of?water?inoculated?at?a?concentration?1:10?dilution?rate?of?the?manufacturer?s?recomended?dose?rate.?Water?
temperature?at?time?of?vaccination?was?18?C.?Salt?was?added?to?the?hauling?tanks?to?raise?chloride?levels?in?the?tanks?to?2?ppt.?Vaccine?was?withheld?from?the?other?half?of?
fingerlings?either?vaccinated?or?non?vaccinated?as?fry.?Thus,?the?experimental?design?consisted?of?four?treatment?groups?(five?replicate?ponds??800?fish/replicate??20,000?
fish/ha)?in?a?2x2?factorial?design:?(1)?non?vaccinated?as?fry?and?non?vaccinated?as?fingerlings,?(2)?vaccinated?as?fry?at?10?d?
post?hatch?and?non?vaccinated?as?fingerlings,?(3)?non?vaccinated?as?fry?and?vaccinated?as?fingerlings,?and?(4)?vaccinated?as?fry?at?10?d?post?hatch?and?vaccinated?as?fingerlings.?
Fingerlings?were?grown?from?early?April?to?mid?November?2005?(225?d).?Ponds?were?managed?using?a?single?batch?cropping?system?and?best?management?practices?as?
described?by?Tucker?and?Robinson?(1990).?Mortalities?were?recorded?throughout?the?experiment.?If?mortalities?were?observed,?the?pond?number,?number?of?mortalities,?date?
of?occurrence,?and?clinical?signs?were?documented.??Fish?suitable?for?necropsy?were?sent?to?the?Southeastern?Cooperative?Fish?Disease?Laboratory,?Auburn?University,?Auburn?
AL,?for?diagnosis.?Fish?were?fed?a?32%?comercial?catfish?diet?as?a?percentage?of?their?body?weight?once?a?day?early??to?mid?morning?six?days?a?week.??The?percentage?body?
weight?fed?was?adjusted?periodically?to?compensate?for?weight?gain,?as?described?by
49?
Robinson?et?al.?(1998).?Daily?feed?allotments?for?each?pond?were?recalculated?weekly,?based?upon?standing?crop?estimates.?Fish?were?fed?to?satiation,?rather?than?as?a?
percentage?of?their?body?weight,?when?temperatures?began?to?drop?in?the?fall.?A?maximum?daily?feed?alotment?of?141?kg/ha?was?established?to?prevent?deterioration?of?
water?quality.?To?ensure?that?al?treatments?had?a?minimal?exposure?to?E.?ictaluri,?na?ve?
fingerlings?exposed?to?E.?ictaluri?were?stocked?into?cages?in?the?experimental?ponds?in?early?June?2005.?A?1.0?L?BHI?culture?of?virulent?E.?ictaluri?was?grown?for?24?h?and?
added?to?a?hauling?tank?with?160?L?of?water?to?expose?fingerlings?via?immersion?for?1?h.?The?concentration?of?the?culture?innoculum?was?8.7?x?10?8?CFU/mL.?One?mesh?cage?
(0.61?m?diameter,?1.07?m?deep,?0.31?m?3?)?attached?to?a?stake?was?placed?in?each?pond.?Exposed?fish?were?stocked?into?each?cage?at?a?density?of?20?fish/cage?for?three?weeks?and?
survivors?then?removed.?Disolved?oxygen?(DO),?temperature,?total?ammonia?nitrogen?(TAN),?pH,?
unionized?ammonia,?nitrite,?chloride,?total?hardnes?(TH??as?CaCO3)?and?total?alkalinity?(TA??as?CaCO3)?levels?were?monitored?throughout?the?course?of?the?experiment.?
Temperatures?and?DO?levels?were?checked?using?a?YSI?550A???oxygen?meter?(Yelow?Springs?Instruments,?Yelowsprings,?Ohio,?USA)?inearly?morning?and?mid??to?
late?afternoon?seven?days?a?week.??Measurements?of?pH?were?performed?using?a?Hach?Sension1???portable?pH?meter?(Hach?Chemical?Company,?Loveland,?Colorado,?USA)?on?a?
weekly?basis?during?the?early?evening.?A?Hach???water?quality?kit?(model?FF?1A)?was?used?to?measure?TAN?and?nitrite?levels?on?a?weekly?basis,?chloride?levels?on?a?bi?weekly?
basis,?and?TH?and?TA?levels?on?a?monthly?basis.?Unionized?ammonia?levels?were
50?
calculated?weekly,?using?pH?levels?and?the?highest?temperature?reached?in?ponds?throughout?the?week.?
Water?quality?parameters?were?modified?as?needed?to?maintain?ranges?conducive?to?good?fish?health.?Disolved?oxygen?levels?were?maintained?above?4.0?ppm?(parts?per?
million)?by?equipping?each?pond?with?a?0.5?hp?pump?spray?aerator?(Aerolator?Systems,?Inc.,?Monroe,?North?Carolina,?USA).?Aerators?were?set?on?a?timer?to?run?daily?from?late?
evening?to?early?morning?(7?h).?Salt?was?added?to?ponds?if?chloride?levels?fell?below?50?ppm?or?ten?times?the?level?of?nitrites.?Powdered?agricultural?limestone?was?added?if?TA?
levels?fell?below?35?ppm.?At?harvest,?fish?were?counted?and?weighed?to?determine?the?final?standing?crop?of?
each?pond.?Survival,?net?production,?feed?conversion?ratios?(FCRs),?and?average?fish?weight?were?calculated.??During?the?study,?standing?crops?estimates?were?calculated?using?
initial?standing?crops?and?estimates?of?FCR,?based?upon?average?fish?size,?provided?by?Robinson?et?al.?(1998).?
Statistical?Analyses?All?statistical?analyses?were?performed?using?Statistical?Analysis?System?version?
9.1.3?software?(SAS?Institute,?Inc.,?North?Carolina,?USA).?For?the?first?laboratory?study,?mean?antibody?titers?(log2)?of?each?treatment?were?subjected?to?a?2?way?ANOVA?
(MIXED?procedure),?with?LS?means?compared?by?t?tests,?to?determine?if?significant?statistical?differences?existed?among?them.?The?second?laboratory?experiment?posed?a?
challenge?because?of?the?presence?of?zeros?in?many?cells,?leading?to?a?highly?skewed?distribution.?Based?on?the?plots?of?studentized?residuals?(histogram?and?quantile?quantile?
plot)?the?data?were?analyzed?with?PROC?GLIMIX?using?lognormal?as?the?distribution
51?
function.?For?the?third?laboratory?study,?percent?survival?for?each?dose?treatment?was?subjected?to?a?1?way?ANOVA?(MIXED?procedure),?with?LS?means?compared?by?t?tests.?
For?the?pond?study,?mean?survival?rates,?FCRs,?net?production,?and?water?quality?parameters?for?each?treatment?were?subjected?to?a?2?way?ANOVA?(MIXED?procedure),?
with?LS?means?compared?by?t?tests.?For?all?analyses,?P???0.05?was?considered?significant.
52?
RESULTS?
Laboratory?Experiment?1?In?the?first?experiment,?with?the?water?temperature?maintained?at?24?C,?antibody?
titers?(log2)?against?E.?ictaluri?ranged?from?0.80??0.46?in?fish?vaccinated?at?2.44?x?10?4?CFU/mL?for?4?h?to?4.40??0.36?in?fish?vaccinated?at?3.16?x?10?6?CFU/mL?for?8?h?(Table?1).?
The?main?efect?of?dose?was?found?to?be?significant.?Mean?antibody?titers?were?significantly?higher?in?fish?vaccinated?at?3.48?x?10?5?CFU/mL?than?in?fish?vaccinated?at?
2.44?x?10?4?CFU/mL.?In?addition,?antibody?titers?were?significantly?higher?in?fish?vaccinated?at?3.16?x?10?6?CFU/mL?than?in?fish?vaccinated?at?the?lower?doses.?The?main?
efect?of?time,?however,?was?not?found?to?be?significant.?No?significant?diference?was?observed?between?the?mean?antibody?titer?for?fish?vaccinated?at?4?h?(2.11)?and?the?mean?
antibody?titer?for?fish?vaccinated?at?8?h?(2.61),?nor?was?a?dose*time?interaction?observed.?Laboratory?Experiment?2?
In?the?second?experiment,?mean?antibody?titers?for?fish?vaccinated?at?3?doses?and?maintained?at?18?C?(log2??95%?confidence?intervals)?were?0.09?(0.0?0.42),?0.35?(0.05??
0.82),?and?1.05?(0.50?1.88)?for?the?low,?intermediate,?and?high?doses,?respectively?(Fig.?1).?No?significant?diferences?in?antibody?titers?were?observed?between?treatments.
53?
Laboratory?Experiment?3?Channel?catfish?fingerlings?were?vaccinated?by?immersion?at?1.95?x?10?4?,?3.80?x?
10?5?,?or?1.95?x?10?6?CFU/mL,?along?with?a?control,?for?4?h,?and?later?challenged?withE.?ictaluri.?Mean?mortality?rates?(??SE)?ranged?from?78.3??2.04%?in?control?fish?to?
33.9??12.28%?in?fish?vaccinated?at?the?highest?dose?(Table?2).?Significant?diferences?were?observed?between?control?fish?and?fish?vaccinated?at?the?highest?dose?and?between?
fish?vaccinated?at?the?lowest?dose?and?fish?vaccinated?at?the?highest?dose.?The?relative?percent?survival?(RPS)?for?fish?(Amend?1981)?vaccinated?at?the?highest?dose?was?56.7?
compared?to?the?control?treatment.? Pond?Study?
Channel?catfish?fingerlings?that?had?been?vaccinated?with?AQUAVAC?ESC?were?grown?out?in?ponds?for?8?mos.?Mean?survival?rates?(?SE)?ranged?from?85.3??1.71%?in?
the?control?fry?control?fingerling?treatment?to?91.3??0.71%?in?the?vaccinated?fry?vaccinated?fingerling?treatment.?Mean?FCR?values?ranged?from?1.38??0.02?in?the?
vaccinated?fry?vaccinated?fingerling?treatment?to?1.42??0.02?in?the?vaccinated?fry?control?fingerling?treatment.?Mean?final?average?weights?ranged?from?0.51??0.02?kg?in?the?
vaccinated?fry?control?fingerling?treatment?to?0.56??0.01?kg?in?the?vaccinated?fry?vaccinated?fingerling?treatments.?Mean?net?production?ranged?from?8,356??380?kg/ha?
in?the?vaccinated?fry?control?fingerling?treatment?to?9,511??163?kg/ha?in?the?vaccinated?fry?vaccinated?fingerling?treatment?(Table?3).??The?main?efect?of?vaccination?at?the?fry?
stage?on?survival?was?found?to?be?significant.?Survival?in?fod?fish?vaccinated?as?fry?(90.18%)?was?3.62%?higher?than?survival?in?fod?fish?non?vaccinated?as?fry?(86.56%).?
However,?the?main?efect?of?vaccination?at?the?fingerling?stage?on?survival?was?not?found
54?
to?be?significant,?nor?was?a?fry?fingerling?interaction?observed.??The?main?efects?of?vaccination?at?the?fry?stage?and?vaccination?at?the?fingerling?stage?on?FCR?values,?
average?weights,?or?net?production?figures?were?not?found?to?be?significant,?nor?were?any?fry*fingerling?interactions?observed.?
Water?quality?parameters?were?monitored?throughout?the?course?of?the?field?study.?Nitrite,?TAN,?pH,?and?chloride?levels?are?reported?for?each?treatment?(Table?4),?however,?
no?significant?diferences?were?observed?for?either?of?the?vaccination?efects,?nor?were?there?any?interaction?efects.?Likewise,?no?significant?effects?were?observed?in?morning?
or?afternon?DO,?total?alkalinity,?or?total?hardnes?levels?for?any?of?the?main?factors,?nor?were?there?any?interaction?efects?(Table?5).??The?mean?(??SE)?number?of?times?morning?
or?afternon?DO?levels?dropped?below?2.5?ranged?from?0.4???0.24?in?the?non?vaccinated?fry/vaccinated?fingerling?treatment?to?1.0???0.55?in?the?non?vaccinated?fry/non?vaccinated?
fingerling?treatment?(Table?6).?The?mean?(??SE)?number?of?times?pH?levels?rose?above?9.5?ranged?from?1.2???0.10?in?the?vaccinated?fry/non?vaccinated?fingerling?treatment?to?
1.8???0.73?in?the?non?vaccinated?fry/vaccinated?fingerling?treatment.?The?overall?change?of?average?morning?and?afternon?water?temperatures?in?the?catfish?ponds?is?displayed?in?
Figure?2.
55?
Table?1.?Mean?antibody?titers?(log2???SE)?to?E.?ictaluri?of?variable?vaccine?(AQUAVAC??ESC)?dose?and?imersion?time?trial?conducted?at?24?C.?Values?within?the?same?row?or?
column?with?diferent?letter?designations?are?significantly?diferent?(P?0.05).?Dose?(CFU/mL)?
Time?(h)? 10?4? 10?5? 10?6? Mean?Pooled?SE?4? 0.80?0.46?2.00?0.54?3.53?0.46?2.11?a? 0.28?
8? 0.93?0.46?2.50?0.64?4.40?0.36?2.61?a? 0.28?Mean? 0.87?a? 2.25?b? 3.97?c?
Pooled?SE? 0.35?0.35?0.35?P?value?dose? <0.0001?
P?value?time? 0.2170?P?value?dose*time?0.756
56?
Figure?1.?Mean?antibody?titers?(log2)?toE.?ictaluriof?fingerlings?vaccinated?with?AQUAVAC?ESC?at?three?diferent?doses?for?4?h?at?18?C.?Diferent?letters?next?to?value?
points?indicate?significant?diferences.
57?
Table?2.?Mean?survival?rates?(?SE)?of?fingerlings?vaccinated?with?AQUAVAC?ESC?for?4?h?at?variable?doses.?Values?within?the?same?row?or?columnwith?diferent?letter?
designations?are?significantly?different?(P?0.05).?Vaccine?dose? Mortality?(%)? RPS?1?
Control? 78.3?2.04?a? ??10?4? 65.0?8.90?a? NS?2?
10?5? 55.3?10.62?a,b? NS?10?6? 33.9?12.28?b? 56.4?
Pooled?SE? 9.31?1?Relative?Percent?Survival?
2?Not?Significant
58?
Table?3.?Mean?survival?rates,?FCR,?average?final?weights,?and?net?production?(?SE)?of?vaccinated?fod?fish?trial.?An?asterisk?indicates?the?P?value?is?significant?at?P?0.05.?A??V??
indicates?vaccination?occurred,?while?a??NV??indicates?no?vaccination.?Survival?
Rate?(%)? FCR? Average?Final?Weights?(kg)? Net?Production?(kg/ha)?Treatments?
Fry??Fingerling?NV?????NV?85.3?1.71?1.41?0.02?0.54?0.03?8,540?632?
V?????NV?88.9?1.91?1.42?0.02?0.51?0.02?8,356?380?NV?????V?87.7?1.44?1.41?0.03?0.56?0.02?9,004?316?
V?????V? 91.3?0.71?1.38?0.02?0.56?0.01?9,511?163?Pooled?SE? 1.51? 0.02? 0.02? 409?
Fry?efect?NV? 86.5? 1.41? 0.55? 8,772?
V? 90.1? 1.40? 0.54? 8,933?Pooled?SE? 1.07? 0.02? 0.02? 290?
P?value? 0.03*?0.76? 0.62? 0.70?Fingerling?efect?
NV? 87.1? 1.42? 0.53? 8,448?V? 89.5? 1.39? 0.56? 9,257?
Pooled?SE? 1.07? 0.02? 0.02? 290?P?value? 0.13? 0.37? 0.15? 0.07
59?
Table?4.?Mean?values?(ppm?except?pH???SE)?of?TAN,?pH,?nitrite,?and?chloride?during?vaccinated?fod?fish?trial.?An?asterisk?indicates?the?P?value?is?significant?at?P?0.05.?A?
?V??indicates?vaccination?occurred,?while?a??NV??indicates?no?vaccination.?TAN?1? pH?Nitrite? Chloride?
Treatment?Fry???Fingerling?
NV????NV?1.32?0.08?8.06?0.11?0.40?0.06?57.70?3.45?V?NV?1.54?0.31?8.16?0.09?0.44?0.04?58.70?1.38?
NV????V? 1.34?0.17?8.06?0.15?0.48?0.14?55.00?2.16?V?????V? 1.62?0.23?7.96?0.15?0.41?0.07?55.14?1.41?
Pooled?SE? 0.22? 0.09? 0.06? 2.26?Fry?efect?
NV? 1.33? 8.06? 0.45? 56.35?V? 1.58? 8.06? 0.43? 56.92?
Pooled?SE? 0.15? 0.09? 0.06? 1.60?P?value? 0.26? 1.00? 0.82? 0.80?
Fingerling?efect?NV? 1.43? 8.11? 0.42? 58.20?
V? 1.48? 8.01? 0.45? 55.07?Pooled?SE? 0.15? 0.09? 0.06? 1.60?
P?value? 0.82? 0.46? 0.73? 0.18?1?Total?Ammonia?Nitrogen
60?
Table?5.?Mean?values?(ppm???SE)?of?morning?and?evening?DO,?total?hardnes,?and?total?alkalinity.?A??V??indicates?vaccination?occurred,?while?a??NV??indicates?no?vaccination.?
DOa?1? DOp?2? TH?3? TA?4?Treatment?
Fry???Fingerling?NV????NV?6.57?0.17?7.30?0.50?72.80?6.44?43.90?2.92?
V?NV?6.82?0.15?7.94?0.53?66.94?4.27?44.50?3.49?NV????V? 6.66?0.12?7.44?0.56?65.48?5.54?41.20?3.89?
V?????V? 6.62?0.15?6.81?0.62?60.10?6.44?34.98?1.27?Pooled?SE? 0.14? 0.55? 5.75? 3.05?
Fry?efect?NV? 6.61? 7.37? 69.14? 42.55?
V? 6.72? 7.38? 63.52? 39.74?Pooled?SE? 0.10? 0.39? 4.06? 2.16?
P?value? 0.47? 0.99? 0.34? 0.37?Fingerling?efect?
NV? 6.69? 7.62? 69.87? 44.20?V? 6.64? 7.13? 62.79? 38.09?
Pooled?SE? 0.10? 0.39? 4.06? 2.16?P?value? 0.72? 0.39? 0.24? 0.06?
1?Disolved?Oxygen?A.M.?2?Disolved?Oxygen?P.M.? 3?Total?Hardnes?as?CaCO3?4?Total?Alkalinity?as?CaCO
3
61?
Table?6.?Mean?number?of?times?(??SE)?disolved?oxygen?fel?below?2.5?ppm?and?pH?rose?above?9.5?in?channel?catfish?ponds?stocked?at?20,000?fish/ha.?
Treatment?Fry??Fingerling?n? Times?of?disolved?oxygen?below?2.5?mg/L? Times?of?pH?above?9.5?
NV???NV?5? 1.0???0.55? 1.6???0.81?V????NV?5? 0.6???0.24? 1.2???0.10?
NV????V? 5? 0.4???0.24? 1.8???0.73?V?????V? 5? 1.0???0.45? 1.6???1.60
62?
Figure?2.?Overal?change?of?average?morning?and?afternon?water?temperature?(?C)?in?the?catfish?ponds?stocked?at?20,000?fish/ha.?
0?5?10?15?
20?25?30?35?
20?May?
05?3?Jun?05?17?Jun?05?1?Jul?05?15?Jul?05?29?Jul?05?12?Aug?05?26?Aug?05?9?Sep?05?
23?Sep?0
5?7?Oct?05?
21?Oct?0
5?4?Nov?05?
Date?
Tempera
ture?(?C)
?
AM?PM
63?
DISCUSSION?
In?the?first?laboratory?experiment,?mean?antibody?titers?were?measured?after?administering?lower?doses?of?AQUAVAC?ESC???for?longer?immersion?times?at?24?C.?
Folowing?administration?of?the?vaccine,?antibodies?specific?for?E.?ictaluri?were?produced,?which?agrees?with?previous?studies.?Serum?antibodies?have?ben?shown?to?be?
present?after?immunization?with?bacterin?(Vinitnantharat?and?Plumb?1992,?1993??Plumb?and?Vinitnantharat?1993??Klesius?and?Sealy?1995??Thune?et?al.?1997??Shoemaker?and?
Klesius?1997)?and?have?ben?shown?to?be?indicative?of?prior?exposure?(Klesius?et?al.?1991??Klesius?1992b??Vinitnantharat?and?Plumb?1993??Antonio?and?Hedrick?1994).?
A?direct?relationship?was?observed?between?mean?antibody?titers?and?vaccine?dose.?Other?researchers?have?observed?a?direct?relationship?between?dose?and?vaccine?
uptake?utilizing?this?vaccine.?Wise?and?Terhune?(2001)?were?able?to?recover?E.?ictaluri?RE?33?from?a?greater?percentage?of?fry?vaccinated?at?2.4?x?10?7?CFU/mL?and?2.5?x?10?6?
CFU/mL?than?fry?vaccinated?at?2.5?x?10?5?CFU/mL.?The?level?of?a?primary?humoral?immune?response?is?usually?corelated?with?vaccine?or?antigen?dose??(Ellis?1988).?
Vinitnantharat?and?Plumb?(1992)?injected?channel?catfish?intraperitoneally?with?killed?E.?ictaluri?at?varying?doses?and?found?fish?exposed?to?2.0?x?10?4?cels/fish?had?lower?antibody?
titers?than?fish?exposed?to?2.0?x?10?6?or?2.0?x?10?8?cells/fish.?Marsden?et?al.?(1996)?measured?antibody?responses?in?rainbow?trout?vaccinated?with?a?mutant?strain?of
64?
Aeromonas?salmonicida?at?doses?ranging?from?2.0?x?10?6?to?2.0?x?10?9?and?found?a?clear?vaccine?dose?effect.?Moore?et?al.?(1998)?imersed?1.5?3.0?g?rainbow?trout,?
Onchorhynchus?mykiss,?for?24?h?in?suspensions?of?1.0?x?10?5?,?1.0?x?10?6?,?or?1.0?x?10?7?Bovine?Serum?Albumin?(BSA)?conjugated?fluorescent?latex?microspheres?per?mL?and?
found?that?uptake?was?proportional?to?suspension?microsphere?concentration.?The?main?efect?of?time?on?antibody?response?was?not?found?to?be?significant?in?
the?first?laboratory?experiment.?To?our?knowledge,?this?is?the?first?report?of?time?being?investigated?as?a?main?efect?with?a?live?vaccine?in?channel?catfish.?Other?researchers,?
however,?have?observed?time?to?have?a?significant?efect?on?antigen?uptake?in?studies?with?rainbow?trout?(Tatner?1987??Moore?et?al.?1998).?The?absence?of?a?significant?effect?
of?time?on?antibody?titers?in?the?present?study?may?have?been?due?to?the?inability?of?extending?time?from?4?h?to?8?h?to?compensate?for?the?dilutions?used.?Tatner?(1987)?
suggested?that,?when?antigen?concentration?is?not?limiting?in?direct?imersion?vaccinations,?only?a?certain?amount?of?antigen?will?enter?a?fish?at?a?given?antigen?
concentration,?regardless?of?immersion?time.??Conversely,?if?a?vaccine?is?diluted?too?greatly,?then?fish?will?quickly?uptake?all?available?antigens,?and?a?time?extension?will?not?
result?in?further?uptake.?In?the?second?laboratory?experiment?conducted?at?18?C,?overgrowth?of?
contaminants?from?the?pooled?water?samples?prevented?the?final?vaccine?doses?in?the?aquaria?from?being?determined.??Because?similar?procedures?for?diluting?were?folowed?in?
the?first?laboratory?experiment,?it?is?assumed?that?final?doses?were?in?the?range?of?1?x?10?4?,?1?x?10?5?,?or?1?x?10?6?CFU/mL.?We?refer?to?the?doses?here?as?low,?intermediate,?and?high.?
Mean?antibody?responses?of?fingerlings?vaccinated?at?18?C?appeared?to?be?lower?than
65?
those?generated?when?vaccination?occurred?at?24?C.?However,?no?direct?comparisons?can?be?made?between?the?two?experiments.?
One?explanation?for?the?lack?of?a?significant?titer?response?lies?in?the?fact?that?the?immune?system?of?warm?water?fish?is?generally?depressed?at?sub?optimal?temperatures?
(Rijkers?1982).?A?rapid?drop?in?water?temperature?from?22?C?to?17?C?has?been?shown?to?result?in?suppresion?of?T?helper?cels?and?lower?B?cel?responses?to?T?dependent?
antigens?(Clem?et?al.?1984??Miller?and?Clem?1984).?However,?acclimation?of?fish?to?17?C?for?longer?than?2?weeks?has?been?shown?to?result?in?recovery?of?T?helper?cell?functionin?
vitro?(Clem?et?al.?1984).?B?cels?isolated?from?channel?catfish?acclimated?for?at?least?two?weeks?at?17?C?showed?significant?responses?to?both?T?independent?and?T?dependent?
antigens,?regardles?of?culture?temperature?(Miller?and?Clem?1984).?In?the?present?study?fish?were?acclimated?to?18?C?for?two?weeks?before?administering?the?vaccine.??The?
experiments?of?Clem?et?al.?(1984)?and?Miller?and?Clem?(1984)?were?conducted?in?vitro,?whereas?the?present?study?was?a?reflection?of?the?immune?response?in?vivo.?Components?
of?the?adaptive?immune?system?of?channel?catfish?may?respond?to?sub?optimal?temperatures?diferentlyin?vivo?thanin?vitro.?
A?second?explanation?for?the?low?antibody?response?observed?in?fish?vaccinated?at?18?C?might?be?that?fish?were?sampled?too?early.?Rijkers?et?al.?(1980)?showed?that?the?
magnitude?of?the?humoral?immune?response?in?carp?in?vivoremained?the?same?at?low?temperatures,?but?the?amount?of?time?required?to?generate?the?response?was?increased.?If?
the?kinetics?of?the?immune?response?in?channel?catfish?are?comparable?to?that?of?carp,?then?sampling?fish?later?than?21?d?post?vaccination?may?have?shown?fish?vaccinated?at
66?
18?C?to?generate?higher?antibody?titers?and?may?also?have?resulted?in?a?positive?corelation?between?antibody?titers?and?dose,?as?we?observed?in?fish?vaccinated?at?24?C.?
In?addition,?future?studies?will?need?to?investigate?whether?fingerlings?vaccinated?at?18?C?are?indeed?protected?against?E.?ictaluri.?Rijkers?et?al.?(1980)?did?not?observe?fish?
vaccinated?at?18?C?to?generate?significantly?greater?secondary?immune?responses.?This?observation?suggests?that?vaccinating?fingerlings?at?18?C?fails?to?stimulate?development?
of?long?lived?memory?cels.?The?delayed?nature?of?the?primary?immune?response?at?18?C?would?obviate?even?the?short?term?protection?aforded?by?higher?levels?of?antibodies?and?
activated?T?cels?in?the?Southeastern?U.S.A.,?where?pond?temperatures?can?rapidly?rise?from?low?temperatures?to?those?conducive?to?E.?ictaluri?infection.?
A?third?explanation?for?the?observance?of?low?antibody?titers?in?fish?vaccinated?at?18?C?may?be?due?to?the?slow?growth?rate?ofE.?ictaluri?folowing?vaccination?at?this?
temperature.?Plumb?and?Vinitnantharat?(1989)?demonstrated?that?optimum?growth?ofE.?ictaluri?occurs?between?25?and?30?C?and?that?litle?or?no?growth?occurs?at?15?C.?Francis??
Floyd?et?al.?(1987)?found?that?percent?mortality?in?channel?catfish?challenged?intraperitoneally?withE.?ictaluriat?concentrations?of?1?x?10?4?,?1?x?10?5?,?or?1?x?10?6?
bacteria/fish?was?significantly?lower?in?fish?challenged?at?17?and?21?C?than?in?fish?challenged?at?23,?25,?and?28?C.?The?researchers?also?observed?that?fish?mortality?was?
corelated?with?inoculation?dose?at?23,?25,?and?28?C,?but?not?at?17?or?21?C.?In?the?third?laboratory?experiment,?channel?catfish?fingerlings?vaccinated?at?the?
highest?dose?had?significantly?greater?survival?than?control?fish?and?fish?vaccinated?at?the?lowest?dose.?Other?researchers?have?found?a?positive?corelation?between?RE?33?vaccine?
dose?and?protection?(Wise?et?al.?2000??Wise?and?Terhune?2001).?Significantly?greater
67?
survival?in?fish?vaccinated?at?the?highest?dose?was?probably?observed?due?to?generation?of?a?larger?specific?immune?response,?as?demonstrated?by?the?first?laboratory?study.?
Antibody?titers?were?used?in?the?first?laboratory?study,?not?as?a?measure?of?the?protection?that?would?be?conferred?folowing?challenge,?but?as?a?measure?of?the?overal?specific?
immune?response?occurring?in?vaccinated?fish?(Klesius?and?Sealy?1995??Plumb?et?al.?1986??Plumb?et?al.?1994??Wolters?et?al.?1996??Thune?et?al.?1997).?The?intracelular?nature?
ofE.?ictalurisuggests?that?cel?mediated?immunity,?working?in?conjunction?with?humoral?immunity,?would?be?important?in?providing?protection?(Abbas?and?Lichtman?2003e),?and?
several?studies?have?shown?this?to?be?the?case?(Shoemaker?and?Klesius?1997??Antonio?and?Hedrick?1994??Sheldon?and?Blazer?1991).?
The?highest?dose?used?in?the?challenge?coresponds?to?a?1:10?dilution?of?the?AQUAVAC?ESC?vaccine.??Protection?against?ESC?may?have?ben?observed?with?the?
highest?dose,?even?though?it?represents?a?dilution?of?the?vaccine,?due?to?the?ability?of?time?to?compensate?for?a?lower?dose.??Standard?protocol?for?administration?of?AQUAVAC??
ESC?cals?for?fish?to?be?vaccinated?for?a?total?of?17?min,?but?fish?were?vaccinated?for?4?h?in?the?present?study.?Other?studies?have?shown?the?ability?of?time?to?compensate?for?
lower?doses.?Tatner?and?Horne?(1985)?observed?that?brown?trout,Salmo?trutta,?vaccinated?and?then?challenged?with?Yersinia?ruckeri?had?significantly?higher?survival?in?
treatment?groups?with?lower?vaccine?doses?and?longer?exposure?times?than?those?treatments?with?higher?vaccine?doses?and?shorter?exposure?times.??Tatner?(1987)?
immersed?3?mo?old?rainbow?trout?fry?in?a?radiolabeled?Aeromonas?salmonicida?for?various?dilution*time?combinations,?and?found?that?certain?combinations?of?time?and?dose?
were??equivalent?,?with?respect?to?the?amount?of?antigen?taken?up?by?the?fish.
68?
Confounding?factors?may?be?responsible?for?the?failure?to?observe?significantly?greater?survival?in?fish?vaccinated?at?the?low?and?intermediate?doses.?The?experiment?
included?only?a?small?number?of?replicates?per?treatment.?Wise?et?al.?(1993)?found?that?a?high?degree?of?innate?variability?is?associated?with?the?immersion?challenge?system,?
limiting?its?ability?to?detect?only?large?diferences?in?treatment?efects?when?smaller?numbers?of?replicates?within?treatments?are?used.??In?addition,?many?of?the?replicate?tanks?
contained?anorexic?fish?and?F.?columnare?was?isolated?from?27%?of?fish?throughout?the?first?week?folowing?challenge,?posibly?having?an?impact?on?overal?results.?
The?eficacy?of?AQUAVAC?ESC?in?fry?has?been?evaluated?in?several?studies?utilizing?both?laboratory?and?field?investigations?(Klesius?and?Shoemaker?1998??Petrie??
Hanson?and?Ainsworth?1999??Wise?and?Terhune?2001??Shoemaker?et?al.?1999??Wise?et?al.?2000??Wise?et?al.?2000??Carrias?2005).?Unpublished?data?(D.?Wise,?Thad?Cochran?
National?Warmwater?Aquaculture?Center,?Stoneville,?MS)?confers?that?comercial?fingerling?operations?can?improve?economic?profitability?through?the?use?of?this?vaccine.?
However,?the?efects?of?this?vaccine?on?fingerlings?grown?to?the?fod?fish?stage?have?not?been?investigated.??Klesius?and?Shoemaker?(1998)?suggested?that?long?term?immunity?is?
posible,?but?field?production?and?economic?data?are?lacking.?In?the?field?experiment?of?this?study?the?main?efect?of?vaccination?at?the?fry?stage?on?survival?of?fingerlings?to?the?
fod?fish?stage?was?found?to?be?significant.?However,?other?production?parameters?were?not?influenced?by?vaccination?at?the?fry?stage,?posibly?due?to?the?relatively?low?number?
of?mortalities?that?occurred?during?the?experiment.?The?main?efect?of?vaccination?at?the?fingerling?stage?on?survival?was?not?found?to?
be?significant.?However,?fish?were?vaccinated?at?18?C?in?the?field?study?and?as?previously
69?
demonstrated?and?discussed,?low?temperatures?may?negatively?influence?the?immune?response?of?channel?catfish?to?E.?ictaluri,?posibly?due?to?suppression?of?immune?system?
components,?low?growth?of?the?bacterium?within?the?fish,?and?les?uptake?of?the?vaccine?(Rijkers?1982??Clem?et?al.?1984??Miller?and?Clem?1984??Plumb?and?Vinitnantharat?1989??
Francis?Floyd?et?al.?1987??Tatner?1987).?Channel?catfish?producers?typically?transfer?fingerlings?into?fod?fish?ponds?during?the?cooler?months,?before?temperatures?rise?above?
20?C,?to?avoid?stressing?the?fish.?In?addition,?they?desire?to?begin?feeding?fish?as?son?as?posible?to?maximize?pond?production?capabilities.?To?try?and?mimic?these?production?
practices,?we?also?chose?to?vaccinate?the?fingerlings?and?transfer?them?to?food?fish?ponds?in?April?just?before?the?start?of?the?new?production?season.?
Another?explanation?for?the?failure?to?observe?significantly?greater?survival?in?vaccinated?fingerlings?and?posibly?even?greater?diferences?in?survival?rates?in?the?fry?
efect?may?be?that?fingerlings?used?for?the?field?study?were?obtained?from?ponds?where?ESC?epizootics?had?occurred?(Carrias?2005).?Studies?have?shown?that?a?primary?
exposure?to?E.?ictaluriresults?in?protection?against?secondary?exposures?(Shoemaker?and?Klesius?1997??Klesius?and?Shoemaker?1997).?In?the?channel?catfish?industry,?ESC?tends?
to?affect?fod?fish?to?a?much?leser?extent?than?it?does?fingerlings,?primarily?because?fod?fish?are?naturally?exposed?to?the?pathogen?in?fingerling?production?phase?(Wise?et?
al.?2004).?Additionally,?a?significant?ESC?outbreak?may?not?have?occurred?in?the?study?ponds.?Although?cages?stocked?with?na?ve?fish?challenged?withE.?ictaluri?were?placed?in?
each?pond?to?introduce?fish?actively?undergoing?an?acute?infection,?there?is?no?way?of?knowing?if?al?the?experimental?fish?were?exposed?to?a?high?enough?pathogen?dose?to?
afect?disease?development.?Mortalities?from?the?experimental?ponds?exhibited?clinical
70?
signs?associated?with?ESC,?but?no?fish?colected?were?suitable?for?necropsy.?Some?mortalities?also?exhibited?clinical?signs?associated?withF.?columnare?infections,?a?
confounding?efect?that?other?researchers?have?noted?in?field?experiments?(Wise?et?al.?2000??Carrias?2005).?Furthermore,?ESC?tends?to?affect?fish?in?ponds?managed?using?a?
single?batch?system,?as?they?were?in?the?present?study,?to?an?even?lesser?extent?because?of?improved?water?quality?and?environmental?conditions?(Tucker?et?al.?1990).?
Sumary?The?results?of?the?laboratory?studies?are?relevant?to?fish?farmers?in?that?it?might?be?
posible?to?increase?survival?in?fod?fish?by?administering?AQUAVAC?ESC?to?fingerlings?at?more?economically?feasible?doses?for?longer?periods?of?time.?Fingerlings?
are?often?transported?from?one?farm?to?another?over?long?periods?of?time,?leaving?open?the?posibility?of?administering?the?vaccine?in?route.??The?ability?to?obtain?the?same?
results?in?exposing?fish?for?4?h?as?in?exposing?fish?for?8?h?is?desirable,?because?a?greater?likelihod?exists?that?fish?will?become?stressed?due?to?remaining?in?hauling?tanks?for?
additional?time.??In?addition,?administering?the?vaccine?for?8?h?may?not?be?practical?on?comercial?fish?farm?operations?for?logistical?and?economic?reasons,?especially?on?farms?
where?fingerlings?and?fod?fish?are?produced?on?or?near?the?same?facility?and?the?number?of?hauling?tanks?and?trucks?are?limited.?
Based?on?these?findings,?the?vaccinations?described?here?in?older/larger?fingerlings?may?have?to?be?restricted?to?fish?being?transported?in?warmer?temperatures.?
Additionally,?the?vaccine?may?have?to?be?restricted?to?a?fish?size?les?than?5?cm,?due?to?the?cost?of?the?vaccine?and?the?total?volume?of?water?used?in?the?vaccination?proces.?
Some?fod?fish?producers?target?this?size?of?fish?for?stocking?directly?into?fod?fish
71?
production?ponds?or??stocker??ponds.?The?inclusion?of?a?stocker?phase?in?single?batch?production?systems?is?becoming?more?comon?in?the?catfish?industry?(Pomerleau?and?
Engle?2003).?This?would?aford?the?opportunity?to?integrate?the?vaccine?as?either?a?boster?or?primary?vaccination?if?the?fish?had?not?previously?survived?an?epizootic.?
The?results?of?the?field?study?are?relevant?to?producers?in?that?purchasing?fingerlings?that?have?been?previously?vaccinated?may?have?economic?benefits.?In?our?
study,?fod?fish?vaccinated?as?fry?were?shown?to?have?3.6%?greater?survival?than?fod??fish?non?vaccinated?as?fry.?Inthe?U.S.?catfish?industry,?fingerling?producers?typically?
charge?as?much?as?$0.004/fish?for?vaccinated?fingerlings,?compared?to?non?vaccinated?fingerlings,?although?costs?vary?less?than?this?depending?on?the?fingerling?operation?(K.?
Shuster,?Intervet,?Inc.).?In?this?study,?fish?were?stocked?at?20,000?fish/ha,?which?would?translate?into?an?extra?$80/ha?for?vaccinated?fingerlings?compared?to?non?vaccinated?
fingerlings.?If?an?average?harvest?weight?of?0.54?kg?and?an?average?market?value?of?$1.54/kg?are?assumed,?as?observed?in?this?study,?then?a?3.6%?increase?in?survival?
translates?into?an?increase?in?net?revenue?of?$519/ha.?To?properly?ases?the?relevance?of?the?field?study?to?U.S.?channel?catfish?farmers,?
however,?it?will?be?necesary?to?conduct?the?study?over?several?years?in?ponds?managed?using?a?multiple?batch?system.?Currently,?most?channel?catfish?farmers?use?multiple??
batch?systems,?rather?than?single?batch?systems,?because?large?standing?crops?are?always?present?and?more?ponds?contain?harvest?sized?fish?throughout?the?year.?However,?
multiple?batch?systems?typically?have?poorer?water?quality?and?are?more?conducive?to?infectious?diseases.?A?study?conducted?over?several?years?using?a?multiple?batch?system
72?
would?be?able?to?take?these?factors?into?account?and,?thus,?more?accurately?evaluate?vaccination?at?the?fry?and?fingerling?stage?under?pond?conditions.
73?
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