32 SEPTEMBER 2013 • WORLD AQUACULTURE • WWW.WAS.ORG response towards a non-completely effective Th 2 response (Bakkemo et al. 2011). Additionally a very weak increase in expression of IL-1β and IL-8 was measured after cod macrophage infection with F. noatunensis subsp. noatunensis. Moreover, F. noatunensis subsp. noatunensis is able to inhibit the respiratory burst in cod leukocytes (Vestvik et al. 2013), a special trait used by intracellular organisms to escape killing in the phagolysosome. Collectively these data present evidence for the capability of F. noatunensis to live in intracellular environments by modulating the host immune system. However, this response can be different in other fish species. In vivo analysis of gene expression of immunerelated genes in zebrafish challenged with Francisella noatunensis subsp. orientalis indicated that zebrafish mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of IL-1β, gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h post-infection and persisting for up to 7 days post-infection (Vojtech et al. 2009). Future work in this area should help in understanding the immunopathogenesis of Francisella in fish. Stimulation of a humoral response with Francisella noatunensis has been achieved in cod and tilapia (Schroder et al. 2009, Soto et al. 2011). Mucosal and serum antibodies play an important role in the adaptive immune response of fish. Antibodies can bind to specific adhesins on the pathogens, agglutinate them and prevent attachment to target cells. They can opsonize the pathogens for phagocytes, as well as serve as an important component in serum, killing by activating the classical pathway of complement, and can potentiate killing by antibody-mediated cell killing. Antibodies play an important role in the prevention of Francisella infection in mammals and fish (Kirimanjeswara et al. 2008, Soto et al. 2011). Antibodies recovered from vaccinated adult tilapia protected tilapia fingerlings during passive immunization when challenged with Fno; however, they were ineffective when high bacterial doses were injected to fingerlings (Soto et al. 2011). As previously stated, stimulation of a strong cell-mediated immune response appears key in the prevention of Francisella sp. infection in higher and lower vertebrates. With this in mind, for killed or sub-unit vaccines to be successful in preventing fish francisellosis, strong adjuvants will probably be needed to stimulate the cell-mediated arm of the immune response. Another possibility is to use live-attenuated vaccines that are able to persist for long periods of time in fish tissues and stimulate an appropriated cell mediated response. In previous work, Soto et al. (2009b, 2011) demonstrated that an insertional mutation in the iglC gene of Francisella noatunensis subsp. orientalis significantly attenuates the bacteria, and that upon immersion challenges, significant protection is given to tilapia fingerlings upon WT challenge. Although these experiments were performed in controlled laboratory settings, it demonstrated that live attenuated vaccines, immersion challenges, and better vaccination practices can potentially be applied in the future to protect fish from this pathogen. Treatment As with potential prophylaxis methods, successful treatment of fish francisellosis depends on an understanding of the pathogenesis and lifestyle of the bacteria. Unfortunately, for Francisella noatunensis very little is known in both areas. Members of the genus Francisella are capable of survival outside the fish host for certain periods of time. However, little is known of potential vectors in water that can maintain viable bacteria in the absence of a suitable fish host. Francisella philomiragia and F. tularensis are capable of invading and replicating in Acanthomoeba (Abd et al. 2003, Verhoeven et al. 2010) and Francisella noatunensis subsp. endociliophora exists as an endosymbiont of the ciliate Euplotes (Schralhammer et al. 2010). These are common protozoa that are found in marine water. The capability of F. noatunensis to survive in other aquatic organisms is unknown. Antimicrobial susceptibility testing methods have been reported for both F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis and are based on methods devised to test F. tularensis (Baker et al. 1985). Broth microdilution testing is performed in a medium consisting of a modified Mueller-Hinton II cation adjusted broth, supplemented with 2 percent IsoVitalex (BD BBL) and 0.1 percent glucose. Both subspecies are susceptible to florfenicol and oxolinic acid and resistant to erythromycin, and trimethoprim/sulfamethoxazole. F. noatunensis subsp. orientalis is susceptible to enrofloxacin, gentamicin, neomycin, oxytetracycline, tetracycline, streptomycin, and nitrofurantoin, but resistant to penicillin, ampicillin, amoxicillin, ceftiofur, sulphadimethoxine, sulphathiazole, novobiocin, tylosin tartrate, and clindamycin (Soto et al. 2012a). However, Isachsen et al. (2012) found F. noatunensis subsp. noatunensis with low susceptibility to oxytetracycline, ciprofloxacin and streptomycin while susceptible to flumequine and rifampin. In the USA, only three antibiotics are currently approved for use in foodfish production. Of these, florfenicol and oxytetracycline are potential therapeutants for fish francisellosis caused by F. noatunensis subsp. orientalis. In the past, mortality events in naturally infected and laboratory challenged tilapia were reduced when fish were treated with oxytetracycline and florfenicol (Mauel et al. 2003, Soto et al. 2010b). Antimicrobials with potential use against fish francisellosis must be given sufficiently early, before granulomata have formed, and have the ability to penetrate eukaryotic cells and act in this environment because of the intracellular lifestyle of the pathogen. With this in mind, early detection and diagnosis of fish francisellosis is of paramount importance for successful treatment. In laboratory-controlled challenges, florfenicol-medicated feed initiated at 1 day post-infection and carried out for 10 days significantly reduced mortalities in tilapia and prevented dissemination of the bacterium to hematopoietic organs such as the spleen, with no pathological changes and reduced numbers of bacteria in the spleen 30 days post challenge. Conversely, medicated feed starting 3-6 days post-infection and administered for 10 days led to the development of a chronic, non-lethal infection, suggesting F. noatunensis subsp. orientalis may have the propensity for latency. This suggests the infection could be contained or eliminated if very early antibiotic treatment (<6 days post-infection) was initiated, preventing the bacterial load from reaching a lethal dose in the host (Soto et al. 2010b). Recently the effects of temperature and salinity on the virulence of F. noatunensis subsp. orientalis in tilapia were investigated (Soto et al. 2012c). Fish maintained at 25° C developed
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