
 flavobacterium psychrophilum , a gram - negative , filamentous , psychrotrophic bacterium belonging to the phylum bacteroidetes , is the causative agent of bacterial cold - water disease ( bcwd ) and rainbow trout fry syndrome ( rtfs ) in freshwater salmonid fish worldwide [ 2 , 3 ] . in chile
, this infectious bacterium was first observed in freshwater aquaculture facilities in 1993 , and the incidence of f. psychrophilum has dramatically increased since then [ 4 , 5 ] . despite the severe impact of this disease , antimicrobial therapies are currently the only method for controlling this condition in farmed fish , and
it has been estimated that 55 tons of florfenicol and oxytetracycline each were used in chilean farms to control outbreaks between 2006 and 2009 .
a tentatively licensed commercial vaccine that contains whole - cells inactivated with formaldehyde , flavomune vaccine ( sag n2160-bp ) , was recently developed in chile .
however it is important to note that its efficacy was tested using injection - based challenge models that completely bypassed the protective functions of the fish skin - mucus layer .
this is significant due to this layer acting as an important barrier to disease infection in fish .
one key for efficient immunization and a powerful generation of specific antibodies is to ensure efficient antigen presentation by cells from the innate immune system .
this event triggers an antigen - specific adaptive immune response that leads to the production of specific antibodies against an invading pathogen .
activated , or noncirculating , leukocytes are involved in the initiation of the adaptive immune response .
recent studies have moreover established various factors that elicit the innate immune response shaping adaptive immunity [ 813 ] .
for instance , splenic neutrophils facilitate the antibody response of marginal zone b cells to microbial antigens .
these granulocytes promote b cell survival , as well as the production of igm , igg , and iga antibodies [ 13 , 14 ] . during the last decade
, the zebrafish ( danio rerio ) has been positioned as a powerful model for immunity research not only for other fish species , but also for mammals [ 1519 ] .
some outstanding characteristics are its genetic tractability and transparency during the embryonic and larval stages , which facilitate monitoring infection and inflammation processes in vivo [ 18 , 2024 ] .
early during zebrafish development , the innate immune system exists in isolation to the adaptive system , which develops later in the larval stages and requires 46 weeks before achieving full functionality . in the present work , zebrafish were used to compare the effect of different f. psychrophilum vaccines on the activation of the innate immune system , using neutrophils as specific innate immune system markers .
these leukocytes are the first cells to be mobilized in response to injury , and they are the first to infiltrate damaged tissue [ 2629 ] . for all assays , the tg(mpx : gfp ) transgenic zebrafish line was used given that it exclusively expresses the green fluorescent protein ( gfp ) in neutrophils and allows tracking individual immune cells in live animals .
zebrafish larvae were bath - vaccinated with three strains of bacteria inactivated by heat ( vah ) or formaldehyde ( vaf ) and with or without an adjuvant .
afterwards , neutrophil migration was determined to investigate the effects of vaccination on the activation of the innate immune system .
two f. psychrophilum strains ( lm-02-fp and lm-13-fp ) were isolated in 2006 in chile from the kidneys of clinically infected rainbow trout ( oncorhynchus mykiss ) for subsequent vaccine preparation .
the lm-02-fp isolate is a sequence type ( st ) 2 strain and antigenically group 1 ( 1150 ) , while lm-13-fp is a st12 strain and group 2 ( 1658 ) [ 31 , 32 ] .
the identity of each isolate was confirmed as f. psychrophilum by using standard phenotyping procedures , including analyses of colony morphology and pigmentation , cell morphology , gliding motility , gram - staining , cytochrome oxidase and catalase activities , oxidation / fermentation reactions , the presence of cell wall - associated flexirubin - type pigments , and the absorption of congo red .
each isolate was routinely grown on a tryptone yeast extract salts medium ( tyes ; 0.4% tryptone , 0.05% yeast extract , 0.02% anhydrous calcium chloride , and 0.05% magnesium sulphate heptahydrate ; ph 7.2 ) in either a liquid or solid state ( tyes supplemented with 1% ( w / v ) agar bacteriological ) .
stock cultures were then frozen and kept at 80c in criobilles tubes ( aes laboratory ) or in a tyes broth with 10% glycerol .
the selected f. psychrophilum isolates were grown in tyes broth for 56  h in order to obtain a final concentration of 3.49  0.51  10 colony forming units ( cfu ) ml .
eight vaccines ( including f. psychrophilum cells and extracellular products ) ( table 1 ) were prepared using the following two inactivation protocols : ( i ) lm-02-fp and lm-13-fp cultures were inactivated by heat at 60c for 6  h and then stored at 4c ; and ( ii ) lm-02-fp and lm-13-fp cultures were inactivated through the addition of formalin to a final concentration of 0.7% and then stored at 4c . the efficiency of each inactivation method was determined by spreading 0.1  ml of culture in 10-fold dilutions ( 10 to 10 in duplicate ) onto a tyes agar plates .
once the plate counts were negative for f. psychrophilum , 1  ml of the culture was taken directly from each inactivation treatment ( vah or vaf ) and seeded onto tyes agar .
aliquots from the vaf or vah vaccines based on lm-02-fp and lm-13-fp , respectively , were mixed 1  :  1 with the adjuvant montanide ims 1312 vg pr ( seppic , france ) according to the manufacturer 's protocols .
in addition to this , the commercial flavomune vaccine ( sag n2160.bp ) , made with two chilean f. psychrophilum strains ( coded 1 and 4 ) , was included for comparative purposes . during the study ,
all embryos were collected by natural spawning , staged according to kimmel and colleagues , and raised in petri dishes at 28.5c in an e3 medium ( 5  mm of nacl , 0.17  mm of kcl , 0.33  mm of cacl2 , 0.33  mm of mgso4 , and without methylene blue ; ph 7.0 ) as previously described . embryonic and larval stadium were expressed in hours post - fertilization ( hpf ) .
fifteen tg(mpx : gfp ) larvae were selected at 48  hpf , as previously described , and bath - vaccinated by incubation with the different vaccines at a 1  :  10 dilution for 1  min ( table 1 ) . a control medium with or without adjuvant
after the bath - vaccination , larvae were washed three times for 5  min with the e3 medium , and neutrophils were monitored .
quantification of neutrophil migration was performed by analyzing the displacement of gfp positive cells outside of the caudal hematopoietic tissue ( cht ) to the region of interest ( roi ) ( figure 1 ) at 2 , 4 , 6 , and 8 hours post - incubation ( hpi ) under a fluorescent stereoscope as previously described .
photographs were taken with a qimaging micropublisher 5.0 rvt camera using the olympus szx16 stereoscope .
images were processed with photoshop cs5.1 , with pictures showing the representative effects of each treatment .
data were analyzed by two - way anova using the prism 6 software ( graphpad software ) .
to analyze the innate immune response triggered by the different vaccine formulations , neutrophil behavior was analyzed in live zebrafish larvae ( figure 1 ) . to quantify the number of mobilized neutrophils , a specific region in the larval tail
was analyzed ( roi ) since neutrophils are normally restricted to the cht , with few circulating cells .
analysis of quantified neutrophil migration indicated that only vah and vaf lm-02-fp vaccines were able to trigger an innate immune response ( figures 2(c ) , 2(d ) , 2(k ) , and 2(l ) ) .
the number of neutrophils detected outside the cht in larvae treated with the vah or vaf lm-13-fp vaccine was the same as that with the tyes medium , the negative control ( figures 2(e)2(h ) , 2(k ) , and 2(l ) ) .
no statistical differences in the number of mobilized neutrophils were found between same - strain vaccines made from either heat or formaldehyde inactivation ( figures 2(k ) and 2(l ) ) . as was expected , when an adjuvant was added to the vaccine formulation all responses considerably increased , even in the tyes medium group ( figures 2(k ) and 2(l ) ; figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/515187 ) .
this result indicates that the adjuvant by itself was able to trigger the innate immune response . in order to determine if vaccine effects changed with time , the same assays as previously described were performed again using one - year - old vaccines .
results indicated that all vaccines had diminished activity ( figure 3 ) . in the case of the vah and vaf lm-02-fp vaccines with an adjuvant ,
a freshly prepared vaccine mobilized an average of 11 neutrophils from the cht at 8  hpi ( figures 2(k ) and 2(l ) ) .
this is in contrast to the old vaccines , which moved approximately 8 neutrophils outside of the cht after 8  hpi ( figure 3 ) .
likewise , the one - year - old vah and vaf lm-02-fp vaccines without an adjuvant showed considerably diminished activity .
moreover , the old vah lm-02-fp vaccine only started to exert effects , as compared to the control , at 6  hpi , while the vaf lm-02-fp vaccine showed effects at 8  hpi ( figure 3 ) .
these results suggest that a year of storage is too long of a time for maintaining efficacy . of significant note , however , is that both vaccines newly formulated with the isolate lm-02-fp triggered an innate immune response at least twice as powerful as that produced by the commercial flavomune vaccine ( figure 4 ) .
vaccination is currently one of the most used practices in the prevention of fish diseases .
therefore , it is very important for the aquaculture industry to know the efficiency and effectiveness of the different commercially available vaccines .
a preliminary step in selecting the most suitable vaccine against a specific pathogen would be to determine its effect on the innate immune system given that the activation of this system could yield a higher production of specific antibodies .
in contrast , antigens unable to activate the innate immune response would probably induce a poor production of antibodies . in chile ,
autochthonous immersion bacterins made from single farm isolates have been used for years as protection against infection by f. psychrophilum , but there is still little information on how effective these antipathogen treatments are in terms of relative survival . moreover ,
the currently available commercial vaccine against f. psychrophilum is insufficient , although there have been numerous studies towards developing a global commercial vaccine . at the present time , there are no comparative studies in fish regarding the responses triggered by formaldehyde or heat inactivated antigens ; only there studies using bath - vaccinations on fish from the same population .
in fact , there are no concordant results in previous works performed with different vaccines containing formaldehyde- or heat - inactivated virulent cells of f. psychrophilum .
this situation could be explained due to experiments being developed in different fish species ( rainbow trout ; coho salmon ( oncorhynchus kisutch ) and ayu ( plecoglossus altivelis ) ) .
moreover , the vaccine formula used in previous studies differed in the content of adjuvant [ 3945 ] .
importantly , madetoja and colleagues   compared the efficacy of formaldehyde- and heat - inactivated antigens in rainbow trout by intraperitoneal injection , demonstrating that both antigens produced high circulating antibody levels and efficient protection against the f. psychrophilum .
the present results agree with this study , with lm-02-fp and lm-13-fp , regardless of the inactivation protocol , showing similar innate immune responses in zebrafish . however , the current study used a set of vaccines supplemented with montanide ims 1312 vg , an aqueous adjuvant containing liquid particles , and an immunostimulating compound listed as a gras substance ( generally recognized as safe ) .
this adjuvant improves the presentation of the antigen to the immune cells and keeps it within the fish tissues , thus increasing the length of protection .
it is important to highlight that it does not cause off - target effects such as melanosis . in this sense ,
the present results demonstrate that montanide ims 1312 vg can be used in studies on fish vaccination against f. psychrophilum . moreover , this adjuvant is able to activate an innate immune response . at present , further immunological studies are in progress in rainbow trout in order to establish the adaptive immune responses to specific antigens ( unpublished data ) .
another factor that should be studied in depth before designing an effective vaccine is the serotyping within the bacterial species .
mata and colleagues   proposed a serotyping system for f. psychrophilum using slide agglutination and compared it to the serological schemes previously proposed by lorenzen and olesen   and izumi and wakabayashi .
three to seven serotypes , mainly associated with the host species , were established . in chile , valdebenito and avendao - herrera   determined the existence of antigenic heterogeneity within f. psychrophilum with four patterns of serological reactions . according to this serological scheme , lm-02-fp corresponds to the group 1 , while the strain lm-13-fp belongs to the minor serological group 4 .
thus , a possible explanation for the differences in the results obtained for lm-02-fp and lm-13-fp on the innate immune response in zebrafish may be the existence of different antigenic group .
therefore , differences in serotype should be determined , and cross protection experiments have to be carried out in order to design a vaccine with broad - spectrum effectiveness . in order to compare effects on the activation of the innate immune system for newly formulated vaccines ( vah and vaf lm-02-fp ) and the commercial flavomune vaccine , the aforementioned assay was repeated and found that both newly formulated vaccines were largely more immunogenic than flavomune vaccine .
it is important to note that this commercial vaccine contains whole - cells from two chilean f. psychrophilum strains inactivated by formaldehyde incubation ( http://www.veterquimica.cl/contenido/vacuna-flavomune548 ) . the assay method applied in the present study permitted the analysis of the initial , yet essential , stage of efficient vaccine selection , which is the activation of cellular machinery that will promote antigen presentation and the innate immune response .
results showed that both vaccines with adjuvants ( strain lm-02-fp and lm-13-fp ) exerted similar effects on the innate immune response , but only the lm-02-fp strain was able to induce neutrophil migration without an adjuvant . from this
, it can be speculated that the vah / vaf lm-02-fp vaccines could induce the generation of specific antibodies against f. psychrophilum .
this isin contrast to theeffects triggered by vah / vaf lm-13-fp vaccines , which produced a nonspecific immune response that was mainly due to the adjuvant .
these results suggest thatthe vah and vaf lm-02-fp vaccines would be a better candidate for vaccine formulation .
finally , by using zebrafish as a model , another important outcome of this work was providing an in depth investigation on the mechanisms that control adaptive immunity in fish .
presently , fish vaccines are primarily developed according to mammalian knowledge about adaptive immunity , a scenario that might not be the most appropriate given the differences between fish and mammalian immune systems [ 5255 ] .
it is currently accepted that fish and particularly zebrafish share similarities with the mammalian immune system [ 19 , 52 , 5658 ] .
for example , both have a comparable set of immune cells [ 5963 ] , and the adaptive immune system is active later during embryonic development , after innate immune system development .
however , there are also differences as compared with mammalian immune responses , such as with the most abundant immunoglobulin in fish being igm , whereas in mammals this is iga [ 53 , 64 , 65 ] . besides this
, several important processes have not yet been described in fish , including antigen presentation and the existence of memory cells . in this regard ,
the different transgenic zebrafish lines with fluorescently labeled immune cells [ 30 , 60 , 61 , 6669 ] allow this teleost fish to act as a  live indicator  of immune processes , with the whole event of interest being monitored in vivo .
the vah and vaf lm-02-fp vaccine induced the strongest innate immune response , even when compared with the commercial flavomune vaccine .
however , after a year of storage at 4c , the capacity of both formulated vaccines to trigger an innate immune response considerably decreased .
finally , the methodology described in this paper , using the zebrafish as fish model , is a suitable approach for analyzing mechanisms that control adaptive immunity in fish , as well pathways of bacterial infection and the innate immune response triggered by different vaccines .
 
 flavobacterium psychrophilum is a gram - negative bacterium , responsible for the bacterial cold - water disease and the rainbow trout fry syndrome in freshwater salmonid fish . at present , there is only one commercial vaccine in chile , made with two chilean f. psychrophilum isolates and another licensed in europe . 
 the present study analyzed neutrophil migration , as a marker of innate immune activation , in zebrafish ( danio rerio ) in response to different f. psychrophilum bath vaccines , which is the first step in evaluating vaccine effectiveness and efficiency in fish . 
 results indicated that bacterins of the lm-02-fp isolate were more immunogenic than those from the lm-13-fp isolate . 
 however , no differences were observed between the same bacteria inactivated by either formaldehyde or heat . 
 importantly , the same vaccine formulation without an adjuvant only triggered a mild neutrophil migration compared to the complete vaccine . 
 observations also found that , after a year of storage at 4c , the activation of the innate immune system by the different vaccines was considerably decreased . finally , new vaccine formulations prepared with heat and formaldehyde inactivated lm-02-fp were significantly more efficient than the available commercial vaccine in regard to stimulating the innate immune system . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions