WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2013 31 less affected, with small foci of necrosis and bacteria-laden macrophages. Granulomatous inflammation in the trunk kidney is relatively mild, largely sparing nephrons. Gills have moderate epithelial hyperplasia with few small granulomas and isolated macrophages filled with bacteria. Atrial endothelial hypertrophy is seen in the heart. Mortality rates on tilapia farms can vary tremendously with water temperature, water quality, and handling stress and can range from 5-60 percent (Soto et al. 2009a). The optimum temperature range for outbreaks of disease on tilapia farms is 20-28° C. The pathogenesis of fish francisellosis was studied after an immersion challenge with wild type Fno. The distribution of bacteria in multiple organs, as well as associated lesion development, was investigated after 3, 24, 48, 96, and 192 h by real-time PCR and histopathological examination. Surface mucus collected 3 h post-infection contained the highest number of Fno genome equivalents (GE). After 96 h, marked increases of WT Fno GE were detected in spleen, anterior kidney, posterior kidney, gill, heart, liver, brain, gonad, and the gastrointestinal tract. Increases in bacterial GE also corresponded to the appearance, size and number of granulomas typical of francisellosis, predominantly in the spleen and anterior and posterior kidney segments (Soto et al. 2013). Culture of the Pathogen Following the discovery that these organisms were in fact Francisella and not Piscirickettsia it became obvious that the bacteria should be culturable provided the proper medium was employed. Based on the literature, various media utilized for the culture of Francisella tularensis have been used for the isolation of Francisella from fish, such as cysteine heart agar with bovine hemoglobin (Soto et al. 2009), cysteine heart agar with 5 percent sheep blood (Mikalsen and Colquhoun 2010), modified Thayer Martin agar (Hsieh et al. 2006), or blood agar with 0.1 percent cysteine + 1 percent glucose (Ottem et al. 2009). The antibiotics Polymyxin B (100 units/mL) and/or ampicillin (50 µg/mL) may be added to the primary isolation media to select against fastergrowing secondary contaminants. Immune Response and Vaccination Very little is known regarding treatment and preventative measures for Francisella spp. infections in fish. As with F. tularensis in mammalian species, it appears that F. noatunensis resides and replicates not only in extracellular environments but also inside eukaryotic cells, especially those of the monocytemacrophage lineage (Soto 2010a, Furevik et al. 2011, Bakkemo et al. 2011, Vestvik et al. 2013). In tilapia head kidney-derived macrophages (HKDM), F. noatunensis subsp. orientalis replicate effectively inside phagocytes through 72 h and are ultimately cytotoxic. In cod, F. noatunensis subsp. noatunensis adheres, penetrates and replicates efficaciously inside cells of the monocyte/ macrophage lineage and to a lesser extent in neutrophils and B-cells (Furevik et al. 2011). Although there have only been a few reports of virulence genes identified for F. noatunensis, with the advent of whole genome sequences (Sridhar et al. 2012, Sjodin et al. 2012), scientific reports in this area are expected to significantly increase in the next decade. Genes present in the F. noatunensis pathogenicity island appear to encode important virulence factors that mediate intracellular survival (Sridhar et al. 2012, Sjodin et al. 2012). The iglABCD operon is important for virulence determination in F. noatunensis subsp. orientalis and necessary for induction of disease and intramacrophage survival in tilapia and zebrafish1 (Soto et al. 2009b, 2010b). As with F. tularensis in mammals, a strong cell-mediated immune response appears key in preventing fish francisellosis. Modulation of the host immune response is a trait used by many successful intracellular or facultative intracellular bacterial pathogens like Francisella tularensis and Yersinia pestis (Hunt et al. 2012, Bliska et al. 2013). Very little is known of the immunopathological response of Francisella in fish. Recently, F. noatunensis subsp. noatunensis LPS was found to stimulate a very modest immune response upon challenge to cod phagocytes. The authors of this paper concluded that this might play a role in subrogation of the immune response stimulation (Bakkemo et al. 2011). Additionally, a high IL-10 response in cod indicated that F. noatunensis subsp. noatunensis could drive a stronger immune (CONTINUED ON PAGE 30) FIGURE 4. Francisella sp. visible as small 0.5 x 1.0 µm coccobacilli in the tissue with Geimsa stain. (Photo: Juan Morales) FIGURE 5. Wet mount (squash prep) of unfixed head kidney showing prominent wellformed granulomas with numerous melanomacrophages.
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