Interaction of Francisella asiatica with Tilapia ( Oreochromis niloticus ) Innate Immunity

Abstract

Members of the genus Francisella are facultative intracellular bacteria that cause important diseases in a wide variety of animals worldwide, including humans and fish. Several genes that are important for intramacrophage survival have been identified, including the iglC gene, which is found in the iglABCD operon in the Francisella sp. pathogenicity island (FPI). In the present study, we examined the interaction of wild-type Francisella asiatica and a ⌬iglC mutant strain with fish serum and head kidney-derived macrophages (HKDM). Both the wild-type and the mutant strains were resistant to killing by normal and heat-inactivated sera. The wild-type F. asiatica is able to invade tilapia head kidney-derived macrophages and replicate vigorously within them, causing apoptosis and cytotoxicity in the macrophages at 24 and 36 h postinfection. The ⌬iglC mutant, however, is defective for survival, replication, and the ability to cause cytotoxicity in HKDM, but the ability is restored when the mutant is complemented with the iglC gene. Uptake by the HKDM was mediated partially by complement and partially by macrophage mannose receptors, as demonstrated by in vitro assays. Light and electron microscopy analysis of the infected macrophages revealed intracellular bacteria present in a tight vacuole at 2 h postinoculation and the presence of numerous bacteria in spacious vacuoles at 12 h postinfection, with some bacteria free in the cytoplasm.Francsiella asiatica and Francsiella noatunensis are recently described members of the genus Francisella (42, 43). Francisella noatunensis isolates were recovered from diseased cultured cod (Gadus morhua) in Norway (42,43). Francsiella asiatica (isolate Ehime-1) was recovered from diseased three line grunt (Parapristipoma trilineatum) in Japan and was the isolate used to describe the new species (31, 43). In the last 5 years the bacterium has caused substantial mortality in tilapia (Oreochromis spp.) and other important warm and cold water species cultured in the United States (including Hawaii);

Roles of Inflammatory Caspases during Processing of Zebrafish Interleukin-1β in Francisella noatunensis Infection

“…1). The zebrafish spleen and kidney are major sources of phagocytes (54,59), the primary cell type infected by Francisella in mammals and fish (11,49). Since in mammals the active form of caspase 1 is secreted from cells (22), culture supernatants and cell pellet fractions were assayed separately.…”

“…For infections, F. noatunensis colonies from 4-day-old CHAH plates were scraped into sterile TSBCD broth (Trypticase soy broth [30 g/liter; Sigma], 0.1% cysteine, 0.2% dextrose), vortexed, and grown overnight at 26°C with shaking (200 rpm). Bacteria were pelleted and diluted in sterile PBS to specific OD 600 (optical density at 600 nm) concentrations corresponding to approximate CFU/ml as previously determined (49). Heat killing of bacterial samples was conducted at 56°C for 1 h. Bacterial cells diluted to the appropriate concentrations in PBS were used to infect zebrafish spleen and kidney single-cell suspensions for 8 h at a multiplicity of infection (MOI) of 50.…”

Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

“…This eventually results in release of the bacteria to the extracellular space through host cell lysis or in the formation of Francisella-containing vacuoles through interactions with the autophagic pathway (19). It has been suggested that F. noatunensis subspecies are also able to escape from phagosomes into the cytosol (20)(21)(22).…”

In Vitro and In Vivo Efficacy of Florfenicol for Treatment of Francisella asiatica Infection in Tilapia

“…Macroscopic and microscopic examination often reveals enlarged internal organs containing widespread multifocal white nodules (13,14,15,17,19,20,29). Moreover, F. asiatica has been found to be resistant to serum killing and can penetrate, replicate, and survive in tilapia head kidney-derived macrophages (THKDM) (31).…”

“…are facultative intracellular pathogens, capable of replicating in macrophages and other various cell types in humans, rabbits, rodents, nonhuman primates, and fish. The bacteria may also exist as endosymbionts of amoebae and arthropods (1,2,7,26,31,34). Francisella asiatica and Francisella noatunensis are two recently described members of the genus that causes piscine francisellosis in a wide variety of fish species (19).…”