Metamorphosis of Ichthyophonus Schizonts Transiting the Gastrointestinal Tract of Experimentally Exposed Rainbow Trout.

Other than the initial infectious cell, schizonts are the only stage of the parasite Ichthyophonus sp. that has been identified in the tissues of a living host, and they are known to initiate new infections when ingested by a suitable host. However, after feeding Ichthyophonus-infected tissue to Rainbow Trout Oncorhynchus mykiss, we observed that once infection was initiated, some schizonts proceeded to develop into several other morphologic forms indistinguishable from those previously described from recently deceased hosts, decomposing infected corpses, and in vitro culture. It appeared that not all schizonts participated in the infection process; some initiated infection, as expected, while others passed into the intestines, where they morphed into multiple cell types (e.g., schizonts, some with partially digested or ruptured capsules, ameboid plasmodia, merozoites, hyphenated cells, and empty capsules). Some of these cells were viable when cultured, but none was infectious to naïve Rainbow Trout when administered by gavage. We posit that (1) not all tissue schizonts are programmed to perform the same function or (2) not all respond similarly to their environment. After consumption by a piscivore, those schizonts that do not initiate an infection do not die but rather metamorphose into different cell types as they transit the gastrointestinal tract and are ultimately released back into the aquatic environment through defecation. The fate of these cells after exiting the host is presently unknown, but they likely represent a segment of the Ichthyophonus life cycle.

Infected Donor Biomass and Active Feeding Increase Waterborne Transmission of Ichthyophonus sp. to Rainbow Trout Sentinels.

The precise nature of Ichthyophonus sp. transmission among wild fishes has eluded description for over a century. Transmission among piscivores is direct, via ingestion of infected prey, but there is also evidence for waterborne transmission between infected and uninfected individuals. Transmission among planktivores is believed to be via a waterborne infectious cell, but definitive proof of this mechanism has not been forthcoming. To explore possible mechanisms of transmission we used Rainbow Trout Oncorhynchus mykiss as a model system and examined the consequence of housing infected donor fish with uninfected (sentinel) fish, without physical contact. We examined two variables linked to transmission: (1) feeding and nonfeeding sentinel fish, and (2) biomass of infected donor fish. Specific-pathogen free sentinel trout were placed in fine-mesh baskets suspended in tanks containing varying numbers of larger Ichthyophonus-infected donor fish and held for 10 weeks, during which time they were examined by in vitro explant culture for the presence of Ichthyophonus. Treatment groups consisted of fed and unfed sentinels housed with infected donors of increasing biomass. After 10 weeks infection prevalence in fed sentinels was significantly higher than in unfed sentinels, and Ichthyophonus was detected earlier in fed fish than in unfed fish. There was no correlation between infection prevalence and donor biomass in fed sentinels, but there was a strong correlation between infection prevalence and increasing donor biomass in unfed sentinels. These data suggest that Ichthyophonus is maintained in wild fish populations by two distinct mechanisms: (1) waterborne infectious cells ingested directly from the water by planktivores, and (2) both infected prey and waterborne infectious cells ingested by piscivores. Received November 13, 2015; accepted February 13, 2016.

Case report: concurrent herpesviral and presumptive iridoviral infection associated with disease in cultured shortnose sturgeon, Acipenser brevirostrum (L.), from the Atlantic coast of Canada.

Approximately 8 weeks after a chlorine insult associated with the city water supply, shortnose sturgeon, Acipenser brevirostrum (L.), from one group presented with small (3-4 mm) irregular foci of cutaneous pallor that involved the dorsocranial integument with progressive ulceration of the nascent lesions. Various bacterial organisms were isolated from the cutaneous lesions, but not from the internal viscera. Histologically, the nuclei of the intralesional and perilesional epidermal cells often exhibited margination of the chromatin that resulted in a homogenous, pale, amphophilic, tinctorial quality of the nucleoplasm consistent with a herpesvirus infection. In addition, rare lamellar epithelial cells were prominently enlarged due to an abundant, dense, basophilic cytoplasm characteristic of an iridovirus infection. Inoculation of cutaneous lesion and kidney, spleen, liver sample pools from affected shortnose sturgeon onto white sturgeon spleen (WSS-2) cell line induced cytopathic effect characterized by syncytia formation. Ultrastructural analysis of infected WSS-2 cells revealed viral particles with a characteristic herpesvirus morphology. Intranuclear hexagonal capsids had a diameter of 95-108 nm, and enveloped particles present in the cytoplasm of infected cells had a diameter of 176-196 nm. This is the first report of a herpesvirus and a possible iridovirus-like infection in shortnose sturgeon.

Molecular characterization of "Candidatus Parilichlamydia carangidicola," a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, "Candidatus Parilichlamydiaceae" fam. nov. (order Chlamydiales).

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported "Candidatus Piscichlamydia salmonis" (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as "Candidatus Parilichlamydia carangidicola" and that the new family be known as "Candidatus Parilichlamydiaceae."

PCR testing for diagnosis of Ichthyophonus hoferi: comment on Hamazaki et al. (2013).

It is our opinion that Hamazaki et al. (2013; Dis Aquat Org 105:21-25) overstate the usefulness of PCR as a field diagnostic technique and underestimate the accuracy and utility of in vitro explant culture. In order for field diagnostic studies to be meaningful they should accurately and dependably identify the infected individuals within a population, both subclinical and clinical cases. Although explant culture, like most techniques, can miss some infected individuals, 'false positives' are impossible, unlike for cPCR based methodologies.

Differential characterization of emerging skin diseases of rainbow trout--a standardized approach to capturing disease characteristics and development of case definitions.

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Reproducible challenge model to investigate the virulence of Flavobacterium columnare genomovars in rainbow trout Oncorhynchus mykiss.

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare genomovars in rainbow trout and other salmonid species.

Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout, Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum.

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F. psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28-day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F. psychrophilum.

Comparative proteomic analysis of virulent and rifampicin-attenuated Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild-type strain (CSF 259-93) with a rifampicin-resistant strain and virulence-attenuated strain of F. psychrophilum (CSF 259-93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL-43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL-43. 2D-PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole-cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259-93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259-93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.

Identification of immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD), and this pathogen has large economic impacts on salmonid aquaculture worldwide. Previously, it was demonstrated that high levels of protection against F. psychrophilum challenge were conferred to rainbow trout, Oncorhynchus mykiss (Walbaum), by immunization with distinct molecular mass fractions of the bacterium, and specific antibodies were correlated with protection. In this study, an immunoproteomic analysis of F. psychrophilum was performed using two-dimensional polyacrylamide gel electrophoresis and Western blotting with serum from fish immunized with high- and mid-molecular mass fractions of the bacterium. Mass spectrometry was used to determine the protein identity, and 15 immunogenic proteins were positively identified following Mascot searches of the F. psychrophilum genome. Based on known function and immunogenicity of homologous proteins in other bacterial pathogens, antibodies specific for several of the identified proteins may be important for protective immunity from CWD. These include outer membrane protein OmpA (P60), trigger factor, ClpB, elongation factor G, gliding motility protein GldN and a conserved hypothetical protein. This work increases the understanding of the protective humoral immune response of rainbow trout against these distinct molecular mass fractions of F. psychrophilum and provides new potential targets for recombinant protein vaccine development.

Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout, Oncorhynchus mykiss (Walbaum).

Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD.

The association between metabolic rate, immune parameters, and growth performance of rainbow trout, Oncorhynchus mykiss (Walbaum), following the injection of a DNA vaccine alone and concurrently with a polyvalent, oil-adjuvanted vaccine.

This research demonstrates a significant increase in routine metabolic rate (RMR) following injection of a DNA vaccine concurrently with a polyvalent, oil-adjuvanted vaccine. The increase in RMR was transient and associated with increased activity of both the non-specific and specific immune responses. Rainbow trout (Oncorhynchus mykiss) were injected with a DNA vaccine (DV), a commercially available polyvalent, oil-adjuvanted vaccine (AV), or the two vaccines in combination and sampled at 203, 305, and 406 days (dd) post-vaccine injection (pvi) for RMR and key immune parameters (serum lysozyme activity, serum neutralization antibody titres). The RMR of fish that received both the DV and the AV was significantly higher at 203 dd pvi, compared to fish from all other treatment groups which included the control, the AV, and the DV groups. The increased RMR corresponded to elevated levels of serum lysozyme activity and an earlier seroconversion of virus-specific neutralizing antibodies. To determine if growth performance was affected by the transient increase in RMR, specific growth rate (SGR), percent daily weight gain (WG), and feed conversion ratio (FCR) were determined at 798, 1204, and 1610 dd pvi. Although fish in all three vaccine groups showed significant increases in SGR and WG at 798 and 1610 dd pvi compared to the control group, the overall weight of the fish was not different at the end of the experiment. In summary, this study shows that concurrent injection of a DV and an AV transiently increases the RMR of rainbow trout and changes the manner in which the immune response occurs, but does not affect the overall growth performance of the fish.

Concurrent injection of a rhabdovirus-specific DNA vaccine with a polyvalent, oil-adjuvanted vaccine delays the specific anti-viral immune response in Atlantic salmon, Salmo salar L.

Vaccines are commonly used in salmonid aquaculture as a method of disease prevention. Although there is a substantial amount of published research regarding the immunological and physiological effects following the injection of different polyvalent vaccines and DNA vaccines, there are no published reports examining the physiological and immunological effects of concurrent vaccine injection, which is the situation encountered in aquaculture. Using key immunological parameters such as lysozyme activity and specific antibody titres we examined the short-term activation of the immune response of cultured Atlantic salmon (Salmo salar L.) following concurrent injection with a traditional, polyvalent, oil-adjuvanted vaccine (AV) and an IHNV-specific DNA vaccine (DV). Our results indicate that different aspects of the innate and adaptive immune responses are influenced in either a positive or negative manner. While concurrent vaccine injection elicited an increase in lysozyme activity, changes in antibody titre (Ab) were antigen specific. The production of anti-Aeromonas salmonicida Abs was significantly greater in the combined vaccine group at 296 degree days post-vaccine injection (dd pvi), while the production of anti-Listonella anguillarum Abs was significantly greater at 106 dd pvi in the combined vaccine group. Of even greater interest was the apparent delay in production of IHNV-specific neutralizing antibodies (NAb) when the DV was injected concurrently with the polyvalent AV. The results indicated that concurrent injection of a polyvalent oil-AV and a DV can be beneficial to the production of antibodies; however, the specific anti-viral response may be delayed.

Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout.

The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of approximately 740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout, Oncorhynchus mykiss (Walbaum).

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD(50) of approximately 3 x 10(7) colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re-challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper-immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund's complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.

Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.

Association of red-mark syndrome with a Rickettsia-like organism and its connection with strawberry disease in the USA.

Red-mark syndrome (RMS), a disease seen mostly in rainbow trout, Oncorhynchus mykiss, is of unknown aetiology. The research presented here indicates the presence of an intracellular bacterium in RMS-affected fish. A positive reaction was observed using immunohistochemistry (IHC) with skin lesions, liver, kidney and spleen of affected fish sampled from several locations within the United Kingdom using two different polyclonal antisera raised against Piscirickettsia salmonis. The same reaction was also seen with a number of different anti-P. salmonis monoclonal antibodies (MAbs). A disease with similar clinical signs to RMS, referred to as strawberry disease (SD), has been reported in the USA. A Rickettsia-like organism (RLO) has recently been associated with SD based on analysis of 16S rDNA sequences. Using the same panel of anti-P. salmonis antibodies used to screen the RMS samples, similar staining was obtained in tissue of SD-affected fish by IHC. A polymerase chain reaction (PCR) using RLO-specific primers was also performed on RMS-affected fish from the United Kingdom, and the samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may be responsible for RMS in the United Kingdom and SD in the USA.

Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media.

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease, but the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial genes of F. psychrophilum differentially expressed in vivo may lead to a better understanding of pathogenesis and provide targets for vaccine development. Therefore, the present study used a proteomic approach to identify and quantify proteins of F. psychrophilum following growth in vivo and under iron-limited growth conditions. As determined by 2D polyacrylamide gel electrophoresis (2D-PAGE), numerous proteins exhibited different spot intensities following culture of the bacterium in vivo, and of these, 20 were selected and identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis and Mascot searches of the F. psychrophilum genome. Eighteen proteins exhibited increased spot intensities in vivo, and these included: several chaperone and stress proteins, gliding motility protein GldN, outer membrane protein OmpH, 2 probable outer membrane proteins (OmpA family), probable aminopeptidase precursor, probable lipoprotein precursor, 3-oxoacyl-[acyl-carrier-protein]-reductase, and several proteins with unknown function. Two proteins exhibited decreased spot intensities in vivo and were identified as ferritin FtnA and outer membrane protein OmpA (P60). Culture of F. psychrophilum in iron-limited media resulted in similar protein spot intensity changes for 6 of the 20 proteins identified following growth in vivo. Results from the present study suggest a role of upregulated proteins in the pathogenesis of F. psychrophilum and these may represent potential vaccine candidate antigens.

Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), with recombinant and DNA vaccines produced to Flavobacterium psychrophilum heat shock proteins 60 and 70.

Flavobacterium psychrophilum heat shock proteins (Hsp) 60 and 70 are highly immunogenic and were therefore investigated as potential vaccine candidates. Recombinant Hsps were purified from Escherichia coli and rainbow trout (Oncorhynchus mykiss) were intraperitoneally injected with phosphate buffered saline/Freunds complete adjuvant (FCA), 8 microg of rHsp60/FCA, rHsp70/FCA or a combination of 4 microg each of rHsp60 and rHsp70/FCA. Antibody responses against recombinant Hsp60 and Hsp70 8 weeks post-immunization were observed, but only fish immunized with rHsp70 exhibited highly elevated antibody levels against F. psychrophilum whole cell lysate. Some cross reactivity occurred, which may have been due to the V5 tag common to both proteins. Protection against F. psychrophilum challenge was not observed in any treatments at 8 weeks post-immunization. To further investigate any protective effect of these proteins, hsps were polymerase chain reaction amplified and cloned into pVAX1. Rainbow trout were intramuscularly injected with 8 microg of pVAX1hsp60, pVAX1hsp70 or a combination of 4 microg each of pVAX1hsp60 and pVAX1hsp70. Antibody responses at 4 weeks post-immunization were low and protection was not observed following challenge at 6 or 10 weeks post-immunization. Although Hsps of F. psychrophilum have been shown to be immunodominant, these antigens do not appear to be good vaccine candidates when delivered alone or in combination.