Francisella infections in farmed and wild aquatic organisms
© Colquhoun and Duodu; licensee BioMed Central Ltd. 2011
Received: 15 July 2010
Accepted: 8 March 2011
Published: 8 March 2011
Over the last 10 years or so, infections caused by bacteria belonging to a particular branch of the genus Francisella have become increasingly recognised in farmed fish and molluscs worldwide. While the increasing incidence of diagnoses may in part be due to the development and widespread availability of molecular detection techniques, the domestication of new organisms has undoubtedly instigated emergence of clinical disease in some species. Francisellosis in fish develops in a similar fashion independent of host species and is commonly characterised by the presence of multi-organ granuloma and high morbidity, with varying associated mortality levels. A number of fish species are affected including Atlantic cod, Gadus morhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo salar; hybrid striped bass, Morone chrysops × M. saxatilis and three-lined grunt, Parapristipoma trilinineatum. The disease is highly infectious and often prevalent in affected stocks. Most, if not all strains isolated from teleost fish belong to either F. noatunensis subsp. orientalis in warm water fish species or Francisella noatunensis subsp. noatunensis in coldwater fish species. The disease is quite readily diagnosed following histological examination and identification of the aetiological bacterium by culture on cysteine rich media or PCR. The available evidence may indicate a degree of host specificity for the various Francisella strains, although this area requires further study. No effective vaccine is currently available. Investigation of the virulence mechanisms and host response shows similarity to those known from Francisella tularensis infection in mammals. However, no evidence exists for zoonotic potential amongst the fish pathogenic Francisella.
Table of contents
- 2.Francisella taxonomy and nomenclature
The fish pathogenic Francisella
The diversity of as yet undescribed Francisella
- 3.The disease/s
Farmed vs. wild fish
Transmission and environmental survival
- 5.Diagnosis and detection of Francisella infections
Selective agar media
Culture temperature for primary isolation
Differential phenotypical identification
Universal PCR combined with DNA sequencing
Specific PCR and LAMP
In situ hybridization
Bacterial pathogenesis and host response
As the aquaculture industry worldwide intensifies and diversifies, it is natural that domestication of new aquaculture species results in recognition of "new" infectious agents and diseases. This has been demonstrated repeatedly over the years. In recent years bacteria belonging to the genus Francisella have "emerged" as serious pathogens of various fish species, both farmed and wild, from various geographical regions worldwide [1–7]. The most recent addition to the list represents the first isolation of a molluscan pathogenic Francisella sp. . Francisellosis associated with aquatic organisms is probably not truly novel. The recent spate of diagnoses may be partially related to the increased awareness of such infections combined with adoption of suitable culture media and the widespread availability of non-culture based molecular detection techniques. However, and for whatever reason, it is clear that Francisella infections in fish are serious and more widely distributed than previously thought just a few years ago. Given the relative recent nature of the discovery of these diseases, much scientific work is currently in progress and many research results remain as yet unpublished. While the present review will restrict reporting of research results in the main to published work, as a measure of necessity, references to unpublished work, manuscripts in preparation and personal communications are occasionally made.
2. Francisella taxonomy and nomenclature
2.1. The fish pathogenic Francisella: nomenclature
Validly published species of the Francisella genus.
Francisella tularensis lineage
Francisella philomiragia lineage
Francisella tularensis subsp. tularensis
Francisella tularensis subsp. holarctica
Francisella noatunensis subsp. noatunensis
Francisella tularensis subsp. mediasiatica
Francisella noatunensis subsp. orientalis
Francisella tularensis subsp. novicida
2.2. The diversity of as yet undescribed Francisella
Although culture of Francisella from the environment is possible [12, 22], it is notoriously difficult. Recent studies of fish microbiota  and environmental samples [11, 12] utilising non-culture based methodology have, however, clearly revealed the existence of significant numbers of as-yet undescribed Francisella and Francisella-related species associated with fish and the environment. A number of gene sequences retrieved from these samples belong to the F. philomiragia lineage and are therefore closely related to currently known fish pathogenic species. An as yet un-cultured endosymbiont of the ciliate Euplotes raikovi has been proposed as a novel subspecies of F. noatunensis i.e. Candidatus F. noatunensis subsp. endociliophora , but this name has not yet been validly published according to the International Code of Nomenclature of Prokaryotes. As nearly all such environmental detections to date have been restricted to aquatic environments, these studies may give some indication of the battery of possible "pathogens" awaiting new aquaculture species.
3. The disease/s
Systemic infections in fish caused by Gram-negative intracellular bacteria refractive to culture on standard laboratory media have been recognized for many years. Such infections have been commonly referred to as either Rickettsia-like (RLO) due to morphological similarities with the true Rickettsia or Piscirickettsia-like organisms (PLO) following the description of Piscirickettsia salmonis. The genus Francisella is in fact relatively closely related and similar both morphologically and in terms of pathogenesis, to Piscrickettsia salmonis. However, as the latter organism and its diseases have been extensively reviewed [26, 27], this genus will not be covered in the present review beyond mention here of two recent and significant developments in Piscirickettsia research i.e. the discovery that this bacterium has a facultatively (not obligatory, as previously considered) intracellular nature and may in fact be cultured on cysteine enriched agar media [28, 29], along with the apparent emergence of a novel Piscirickettsia species causing disease in muskellunge, Esox masquinongy and yellow perch, Perca flavescens. Despite morphological similarities, the genera Francisella and Piscirickettsia belong to the γ-proteobacteria and are therefore only distantly related to the true Rickettsia (α- proteobacteria).
While the "agent of tularemia" presumably F. tularensis, was related to infections in fish as early as 1970, this bacterium has not been associated with fish disease in later years . In light of the recent description of the fish pathogenic species, which share a number of phenotypic traits with F. tularensis, it might be speculated that these early detections may have been a case of misidentification. An outbreak of water borne tularemia associated with crayfish fishing in Spain could not be attributed to the crayfish themselves . The "modern" emergence of francisellosis probably started with the identification of a Rickettsia-like organism (RLO) in diseased tilapia farmed in both fresh and saltwater in Taiwan , which is probably the Francisella-like organism described in Taiwanese tilapia by Hsieh et al. . Francisellosis was subsequently identified in farmed tilapia in Latin America , more specifically Costa Rica [19, 34] and several states in mainland USA , while a similar disease associated with a PLO in farmed tilapia in Hawaii , is as yet unconfirmed as francisellosis. The bacterium has additionally been isolated from tilapia in Indonesia  and recently confirmed in tilapia farmed in recirculated systems in England . Other species affected include hybrid striped bass, Morone chrysops x M. saxatilis in california  and three-lined grunt, Parapristipoma trilinineatum in Japan (imported from China) . Other RLO infections which could conceivably be related to Francisella spp. include the RLOs reported from ornamental blue-eyed plecostamus, Panaque suttoni and dragonet, Callionymus lyra. However, it should not be assumed that all RLO/PLO are in fact Francisella spp. The Piscirickettsia salmonis-like organism reported from cultured grouper, Epinephelus melanostigma in Taiwan , in contrast to the confirmed Francisella infecting tilapia  reacted positively with polyclonal anti-P. salmonis sera and may therefore be more related to Piscirickettsia than Francisella.
3.1. Differential diagnoses
Several bacterial diseases may present in a similar manner to francisellosis. Piscine mycobacteriosis, commonly characterised by macroscopically visible multi-organ granuloma caused by a diverse range of different Mycobacterium spp. has been identified in a large number of cultured and wild fish species around the world . Mycobacteria can be refractive to culture and are not always readily observable in histological preparations even when Ziehl-Neelson stained. Nocardia spp. infections may also present in a granulomatous form . Piscirickettsia salmonis infections, which may also present in a similar fashion to francisellosis is most commonly associated with salmonid fish species, yet has been identified in an increasingly diverse range of fish species e.g. European seabass, Dicentrachus labrax and white seabass, Atractoscion nobilis. One of the most common systemic bacterial infections affecting populations of farmed cod in Norway is atypical furunculosis , caused by atypical isolates of Aeromonas salmonicida. This type of infection can result in a disease presenting macroscopically very similar to francisellosis. Although both diseases result in extensive granuloma development, they are quite readily differentiated by histological examination. Mixed infections with F. noatunensis and atypical A. salmonicida and/or Vibrio anguillarum are also relatively common .
3.2. Farmed vs. wild fish
Many systemic bacterial diseases result in relatively rapid death of the affected fish, which disappear from the population and are therefore difficult to detect at low prevalence in wild fish populations. The chronic nature and lengthy course of francisellosis, particularly in cold water marine species such as cod, probably mean that the likelihood of detection of francisellosis in wild fish is more likely than with other Gram-negative infections. Francisellosis is, however, a relatively recently recognised disease, and reports from wild fish are as yet relatively rare. A prevalence of approximately 20%, based on macroscopic observations, was identified in a single year class of wild cod captured off the Swedish west coast in 2004 . Farmed Atlantic cod in Norway are held in net cages in close contact with wild fish (including wild Atlantic cod) which congregate around these structures. A recent screening  of farmed and wild cod as well as other species of fish caught around the Norwegian coastline using Real Time PCR, reported the relatively widespread presence of F. piscicida (a.k.a. F. noatunensis) in wild cod (prevalence 7-11%), from both areas with and without cod farms, although fish exhibiting clinical signs of disease were rare. Unfortunately the disease/infection status in wild fish prior to recent outbreaks in farmed cod is not known and little is understood of the effect of infection pressure from farmed fish to wild fish in these areas. Low levels of infection were also identified in several marine fish species i.e. coalfish, Pollachius virens, pollock, Pollachius pollachius, mackerel, Scomber scombrus, European plaice, Pleuronectes platessa and megrim, Lepidorhombus whiffiagonis and other aquatic organisms such as blue mussels, Mytilus edulis and edible crab, Cancer pagurus. However, the significance of these low level detections is difficult to estimate, considering the extreme sensitivity of the assay and that samples were collected mainly in the proximity of affected cod farms. A low prevalence of PCR positive fish in populations of migratory cod (spawning migration from the Barents Sea) caught off the Lofoten archipeligo in Northern Norway has also been reported . That clinical francisellosis is a disease of long standing in nature has been established in a retrospective study utilising paraffin-embedded samples performed in our own laboratory, which confirmed the existence of francisellosis in wild cod in the North Sea during the 1980s . There are no published reports of francisellosis caused by F. noatunensis subsp. orientalis in wild fish.
3.3. Host specificity
Little information is available relating to specificity of the various Francisella species for the various species of fish from which they are most commonly isolated. F. noatunensis subsp. orientalis (or very closely related bacteria), most commonly isolated from tilapia does, however, cause disease in a number of other fish species including three-line grunt  and a variety of ornamental cichlids , while experimental infections following intraperitoneal injection of F. noatunensis subsp. orientalis have been established in red sea bream, Pagrus major and zebrafish, Danio rerio. That a dose equivalent to 23 cfu was capable of causing mortality in tilapia  while a much higher dose of 3.45 × 105 cfu was required to cause very low mortality in zebrafish  indicates a degree of host specificity at least under the experimental conditions. The virulence of F. noatunensis subsp. noatunensis isolated from Atlantic salmon  and cod for other species of fish is as yet untested or at least undescribed in the literature. Although the total numbers of wild fish other than cod studied by Ottem et al. , were low, generally higher numbers of F. noatunensis were identified in wild cod than non-cod species. Ottem et al.  also reported finding significant levels of F. noatunensis subsp. noatunensis in one farmed Atlantic salmon by quantitative PCR, yet no clinical sign of disease in salmon has been identified in Norway, despite regular surveillance in large, dense populations of salmon farmed in the immediate vicinity of infected cod populations. This, together with the fact that only cod were identified displaying clinical signs of disease in the Swedish epizootic , may indicate that the north Atlantic strain of F. noatunenis subsp. noatunensis has an affinity for Atlantic cod greater than for other species of fish. The Francisella sp. pathogenic for giant abalone, Haliotis gigantea described by Kamaishi et al.  while also virulent in the Japanese black abalone, Haliotis discus discus and identified as the presumptive agent of disease in Yezo abolone, Haliotis discuss hannai, is apparently unable to cause disease in the teleost red seabream.
3.4. Zoonotic potential
While there is some strain dependent variation, F. tularensis is widely recognised as a highly virulent zoonotic agent. F. philomiragia, with which the fish pathogenic species are relatively closely related, also poses a slight, but real zoonotic potential, particularly in individuals with suppressed immunity [56–58]. While both F. tularensis and F. philomiragia are capable of laboratory growth at 37°C, none of the fish pathogenic species are capable of growth at this temperature. Mikalsen et al.  tested pathogenicity of F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis in mice by intraperitoneal injection of relatively high doses of bacteria (5-7 × 107 cfu), without any adverse reaction, disease or subsequent re-isolation of bacteria from internal organs. Thus, laboratory-based evidence would suggest that it is highly unlikely that fish pathogenic Francisella pose a risk of zoonotic infection. It is probably relevant in this context to consider F. noatunensis subsp. orientalis more closely. Of the fish pathogenic Francisella species described to date, this bacterium has the highest optimal and maximum growth temperature and has most commonly been identified in tilapia around the world. Tilapia possess fin spikes which often cause skin injury during handling and/or preparation and such skin injuries may be associated with transmission of zoonotic infections e.g. Streptococcus iniae. That many hundreds of thousands of tilapia infected with F. noatunensis subsp. orientalis must have been handled, processed, prepared and eaten during the last decade/s, without a single case of associated disease being reported, probably constitutes the most compelling "evidence" for lack of zoonotic capability in this group of bacteria.
4. Transmission and environmental survival
Members of the Francisella genus are non-motile and are "transmitted by direct contact with infected animals, through contaminated water or food, or by vectors such as biting insects" . Transmission of francisellosis in fish has an obvious connection with the aquatic environment, and the disease has been identified in both fresh and marine waters [1, 3, 7, 13, 38]. It would appear that francisellosis is highly transmissible under optimal environmental conditions as prevalence of infection within affected stocks of farmed Atlantic cod and tilapia can be extremely high [7, 33] although there is some evidence (Colquhoun, unpublished results) that francisellosis transmission in cod may be reduced at lower temperatures. Tularemia i.e. F. tularensis is known to have a very small least infectious dose of 10 bacterial cells or less . This trait appears to be shared with fish and mollusc pathogenic Francisella, as few as 1 - 23 cfu F. noatunensis subsp. orientalis injected intraperitoneally were capable of causing disease in tilapia while 32 cfu of the abalone pathogenic Francisella sp. described by Kamaishi et al.  killed 100% of intramuscularly injected abolone within 16 days of infection. While the minimum infectious dose for F. noatunensis subsp. noatunensis in cod has not been established, laboratory trials have confirmed the rapid transmission and chronic course of disease in cod [6, 59]. Fish to fish contact is unnecessary and cod may be directly infected via effluent water from tanks containing infected fish (M. Schrøder, pers. comm.). In a cohabitant challenge performed at 12°C, all cohabitant Atlantic cod sampled after 38 days were infected  and by the end of the five month cohabitation period, 100% of cohabitant fish displayed severe macroscopic signs of disease and were culture positive. Interestingly few fish died during this period. Not surprisingly, water temperature appears to play a significant role in development of francisellosis. Progression of infection, transmission and mortality associated with F. noatunensis subsp. noatunensis in cod is low at the lower end of water temperature at which cod may be farmed (< 4°C), although bacteria may also be readily cultured from infected fish during the winter months (Colquhoun, pers. obsv.). The course of disease increases with water temperature up to the maximum temperature at which cod may survive (approaching 20°C). Infection and transmission of F. noatunensis subsp. orientalis appears restricted to 20-28°C in hybrid striped bass  and greater mortality was identified at 15°C than at 30°C in tilapia . Salinity does not seem to have a significant role in disease development as F. noatunensis subsp. noatunensis has been identified in marine farmed Atlantic cod  and in Atlantic salmon farmed in freshwater , while F. noatunensis subsp. orientalis has been isolated from hybrid striped bass and tilapia in fresh water [37, 62] and three-lined grunt in seawater [3, 38]. In the previously mentioned laboratory trial, F. noatunensis subsp. noatunensis could be cultured from the gut of 50% of cohabitant Atlantic cod at termination of the trial , which may indicate the fecal-oral route as an important route of transmission. Identification of F. noatunensis subsp. noatunensis in Atlantic cod eggs may also indicate that the disease can be transmitted vertically, although, this needs to further examined . There is evidence that F. tularensis persist in a viable but non-cultivable (VBNC) state in cold water . Duodu and Colquhoun  found F. noatunensis to enter the same state after 30 and 16 days at 8°C and 12°C, respectively. Although metabolically active, the VBNC fish pathogenic Francisella (in common with F. tularensis) were non-virulent, at least under the experimental conditions tested. It may be that the conditions for revival of virulence were simply not met. A reservoir in aquatic protozoans has been proposed .
5. Diagnosis and detection of Francisella infections
5.1. Macroscopic examination
While severely affected populations often show a high rate of morbidity, from field experiences in Norway it is clear that the disease may become highly prevalent prior to noticeable change in fish appearance or behaviour. Initial clinical signs (in severely affected fish) include emaciation, dark colouration and raised haemorrhagic nodules [6, 7] or skin ulceration  may be observed. Internal macroscopically visible changes are dominated by the multi-organ granuloma development described previously.
5.2. Histological examination
Histological examination of formalin-fixed paraffin embedded tissues (FFPE) is one of the most commonly used diagnostic procedures in fish disease investigation. The histological picture, at least for those species of affected fish for which histological investigations are described, appear to be similar [2, 4, 5, 7, 34, 38, 55]. They show extensive granulomatous inflammation with multiple granulomas , many of which may be liquid-filled . Cells within the granuloma are dominated by hypertrophied foamy macrophages [5, 7], fibroblasts and leukocytes . Granulomas may display a necrotic core [5, 6]. Focal to diffuse necrosis and necrotising vasculitis in affected organs, accompanied by infiltration of mononuclear cells and granuloma formation were described by Mauel et al. . Few or no bacteria may be observable particularly in cases of advanced disease with extensive mature granuloma . Such lesions may be observed in almost any organ or tissue type including the meninges in severe infections .
Media used for isolation of Francisella spp. from fish.
Francisella noatunensis subsp. noatunensis
Atlantic cod Salmo salar
cysteine heart agar + 5% ovine blood
Olsen et al. 
Francisella noatunensis subsp. noatunensis
Atlantic salmon Salmo salar
cysteine heart agar + 5% ovine blood
Birkbeck et al. 
Francisella noatunensis subsp. orientalis
Tilapia Oreochromis sp.
cysteine heart agar + 5% ovine blood
Mikalsen et al. #
Francisella noatunensis subsp. orientalis
Three-lined grunt Parapristipoma trilinineatum
cysteine heart agar + 1% haemoglobin
Kamaishi et al. 
Tilapia Oreochromis sp.
Hsieh et al. 
Tilapia Oreochromis sp.
modified Thayer-Martin agar, selective cysteine heart agar + bovine haemoglobin, selective cystein heart agar + rabbit blood
Soto et al. 
5.3.1. Selective agar media
5.3.2. Culture temperature for primary isolation
Optimal culture temperature differences exist between F. noatunensis subsp. noatunsis, F. noatunensis subsp. orientalis and the molluscan pathogenic strain, which probably reflect evolutionary differences related to host species and environment. Soto et al.  described optimal growth of F. noatunensis subsp. orientalis (putative) at 28°C, while Mikalsen et al.  described optimal growth of F. noatunensis subsp. noatunensis at 22°C. While both types of bacteria are capable of growth at 30°C, F. noatunensis subsp. noatunensis grows poorly at this temperature . That F. noatunensis subsp. noatunensis was also reported as having an optimum temperature of 15-19°C and was unable to grow at 30°C , suggests that agar composition may be important in relation to growth at various temperatures. Kamaishi et al.  reported optimal growth of Francisella sp. from abalone at temperatures between 17 and 22°C. Suitable culture temperatures for isolation of fish and mollusc pathogenic Francisella would therefore generally appear to be in the range of 22°C-25°C.
As Francisella spp. may be cultured on cell-free laboratory media, the benefits of culture in cell-culture may be dubious given its' technically demanding and laborious nature. However, successful cell-culture of Francisella noatunensis subsp. noatunensis has been reported in salmon head kidney (SKK-1) and Atlantic salmon kidney (ASK) cells with best growth in SHK-1 cells  and Francisella sp. (most probably F. noatunensis subsp. orientalis) isolated from tilapia was successfully cultured in chinook salmon embryo (CHSE-214) cells .
5.5. Differential phenotypical identification
Francisella spp. are generally rather biochemically unreactive and the number of phenotypical tests useful for differentiation of the various member species are few. Fish pathogenic Francisella species and F. tularensis share a requirement for cysteine in culture media, and the fish pathogens may thus be initially more easily confused with this species than with their phylogenetically closer relative F. philomiragia which grows quite happily on blood agar. While several commercial kits have been used for phenotypical profiling of fish pathogenic species, reactions may be weak and difficult to interpret , and published comparisons have not included F. tularensis or F. novicida. Fish pathogenic isolates may, however, be fairly rapidly differentiated from F. tularensis and F. novicida by their lack of growth on suitable media at 37°C [19, 20]. Further the fish pathogenic Francisella may be readily distinguished from F. philomiragia (environmental and mammalian isolates) by their requirement for cysteine in culture media and their inability to grow at temperatures of 35°C or above and lack of production of cytochrome oxidase .
5.6. Molecular identification
5.6.1. Universal PCR combined with DNA sequencing
A common theme to most, if not all initial confirmations of francisellosis, is utilisation of the polymerase chain reaction (PCR) in association with "universal" primers directed at the bacterial 16S rRNA gene. Following amplification and DNA sequencing, identification of Francisella-related 16S rRNA gene sequences within tissue samples allows directed culture with appropriate media. Such a strategy was used in identification of the aetiological agent of francisellosis in Atlantic cod [6, 7] Atlantic salmon  tilapia [2, 5], hybrid striped bass , three-lined grunt  and abolone . Isolation and culture of the bacterium has then allowed phenotypical and genetic characterisation, which has in turn provided a basis for both phenotypical based identification and development of specific molecular assays for detection of the respective bacteria within fish tissues.
5.6.2. Specific PCR and LAMP
PCR/Real time PCR/LAMP primers (and probes) used for detection/characterisation of Francisella spp.
Target gene or region
Primer sequence (5' - 3')
Probe sequence (Real time PCR)
F. noatunensis subsp. noatunensis + subsp. orientalis
Ottem et al. 
F. noatunensis subsp. noatunensis + F. philomiragia
Ottem et al. 
F. noatunensis subsp. orientalis
Soto et al. 
Hsieh et al. 
Forsmann et al. 
Barns et al. 
F. noatunensis subsp. noatunensis
Primer set 4 (LAMP) F3
Caipang et al. 
5.6.3. In situ hybridisation
In situ probes used for visualisation of Francisella spp. in aquatic animals.
Primers or probes (5' - 3')
PCR product (286bp)
Hsieh et al. 
PCR product (1113bp)
Hsieh et al. #
Francisella noatunensis subsp. orientalis
Kamaishi et al. 
Kamaishi et al. 
Minimal Inhibition Concentrations.
a.k.a. F. noatunensis subsp.
7. Bacterial pathogenesis and host response
F. tularensis, as a serious zoonotic agent and candidate for biological warfare/terrorism is by far the most significant member of the genus in terms of human impact. While a considerable body of information relating to pathogenesis, virulence and host response (reviewed by Pechous et al.  is available for this bacterium, much relating to the mode of action and genetic basis for virulence remains poorly understood. Although similar work on the fish pathogenic Francisella species is limited, the results generated so far are generally consistent with those from studies focusing on mammalian pathogenic Francisella spp. Homologs of genes associated with virulence in F. tularensis have been identified in F. noatunensis subsp. orientalis, including genes (iglA - D) associated with the type 6 secretion system present on the F. tularensis pathogenicity island. Soto et al.  found that while iglC played no role in protection from serum killing, a functional iglC gene is necessary for intra-macrophage survival. Serum complement and host cell mannose receptors were also identified as important for macrophage internalisation of F. noatunensis subsp. orientalis cells. Zebrafish infected intraperitoneally with F. noatunensis subsp. orientalis displayed a tissue-specific proinflammatory response , with upregulation of inter-leukin-1β (highly specific to viable bacteria), gamma interferon and tumour necrosis factor alpha, 6 h post infection and lasting for up to 7 days.
No commercial vaccine is currently available against Francisella infections in fish, although several vaccine companies are involved in development work in relation to francisellosis in tilapia and cod. Development of a vaccine providing satisfactory protection toward fish pathogenic Francisella spp. may be challenging as observed with other intracellular bacterial pathogens such as Renibacterium salmoninarum and Piscirickettsia salmonis. Several trial vaccines against francisellosis in cod, based on simple whole-cell based preparations (bacterins) have been tested both in experimental challenges and in the field in Norway. None have yet awarded a significant or satisfactory degree of protection. Work contributing to a better understanding of immunological activity and bacterial factors involved in the disease is as yet limited, but includes characterisation of the lipopolysaccharide and β-glucan of Francisella "victoria" (isolated from tilapia, almost certainly F. noatunensis subsp. orientalis)  and identification of a strong, specific antibody response to a 20-KDa non-protein constituent (probably LPS) of F. noatunensis subsp. noatunensis in cod . While a recombinant approach may, as in P. salmonis, offer the promise of increased protection, it may be worth considering the fact that no vaccine against F. tularensis infection in humans is as yet available , despite the greater knowledge of pathogen-host interactions for this disease. Rohmer et al.  proposed that due to the intracellular nature of these bacteria, a live (attenuated) vaccine instead of a component vaccine may be the best approach for successful vaccination. Identification of complete attenuation of F. noatunensis subsp. orientalis by mutation of the iglC* gene as described by Soto et al. , should provide an interesting foundation for further vaccine development.
Infection models, including intraperitoneal-, bath- and cohabitant- challenges exist for F. noatunensis subsp. orientalis[54, 55] and F. noatunensis subsp. noatunensis[6, 59]. Such models are an essential part in vaccine developmental work and batch testing. However, current standards for evaluation of effectiveness of fish vaccines rely on differences between relative percentage survival (RPS) in vaccinated and unvaccinated fish. This may be an effective method of evaluation of protection awarded against systemic bacterial infections normally causing acute mortality episodes e.g. various Vibrio infections, but may be questionable as a means of evaluating a disease like francisellosis which is normally associated (particularly in coldwater species) with a chronic infection. There is a risk that while vaccinated fish may survive the initial exposure and observation period, they may remain infected and the onset of disease merely delayed.
9. Concluding remarks
Despite previous recognition of the disease, the aetiological agents of francisellosis were not identified until recently. As these bacteria are not always readily observed histologically and cannot be cultured in the laboratory media used in routine fish disease investigations, it is likely that diseases caused by this group of bacteria remain under-diagnosed. Improved molecular/genetic tools for specific detection and diagnosis of francisellosis have been developed by a number of groups, but these studies are by no means complete since there remain major gaps in our understanding of the epidemiology and pathogenesis of the bacteria. We are not sure of their life cycle and the mechanisms by which they might spread in the environment. Evidence also exists for the existence of a large number of related bacteria in the environment. There is no doubt that as wild fisheries decline and our dependence on aquaculture products expands, domestication of new species will most probably result in identification of new species and strains of Francisella pathogenic for these species. Development of effective generic vaccines against francisellosis in fish should therefore be a research priority.
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