Intracellular Replication of Edwardsiella ictaluri in Channel Catfish Macrophages

Abstract

The ability of Edwardsiella ictaluri to enter, survive, and replicate in head kidneyÀderived macrophages from channel catfish Ictalurus punctatus was investigated in vitro. Primary macrophage cultures were exposed to E. ictaluri and were sampled after 0 (90 min postinfection), 4, 8, and 12 h. The data indicated that bacterial numbers for E. ictaluri increased 2.6-, 5.1-, and 7.1-fold after 4, 8, and 12 h of incubation, respectively. Microscopic examination of infected cell cultures revealed numerous intracellular bacteria that were contained within clear, spacious vacuoles. Opsonization with normal serum resulted in significantly greater internalization of E. ictaluri at 0 h compared with pretreatment with heat-inactivated serum or no serum at the same time point, and bacteria pretreated with normal serum maintained significantly greater numbers than those in the other treatments after 4 and 8 h. Opsonization, however, did not affect the ability of E. ictaluri to replicate once the cell was entered, as there were statistically significant increases in bacterial numbers at all time points regardless of the serum pretreatment.

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

“…The ability to survive intracellularly is crucial for several bacterial fish pathogens after invasion of their eukaryotic target cells (16,22,41). Distinct subpopulations of macrophages derived from goldfish (Carassius auratus) kidney leukocyte cultures were previously characterized.…”

“…Effective internalization by both cell types was partially mediated by a heat-sensitive serum component, presumably complement. Complement and/or complement receptors (CR) have been associated with efficient internalization of many mammalian and fish pathogens, including F. tularensis, Mycobacterium spp., Listeria monocytogenes, and Edwardsiella ictaluri (6,12,13,16,22). With the F. tularensis live vaccine strain (LVS), optimal phagocytosis by dendritic cells (DC) is dependent on complement factor C3-derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18) (12).…”

“…An optimal medium for culture of tilapia macrophages was designed based on previously published media for cultivation of channel catfish and hybrid striped bass macrophages (16,22) but with an osmolality of 320 mosmol/kg H 2 O to match tilapia serum osmolality. The complete tilapia macrophage medium (CTMM) consisted of Roswell Park Memorial Institute (RPMI) medium 1640 (GIBCO, Invitrogen Corp., Carlsbad, CA) with 14 mM HEPES buffer (GIBCO, Invitrogen Corp.), 0.3% sodium bicarbonate (GIBCO, Invitrogen Corp.), 0.05 mM 2-beta-mercaptoethanol (Sigma Chemical Co., St. Louis, MO), and 5% heat-inactivated, pooled tilapia serum.…”

“…Previous protocols were modified for the collection and culture of tilapia macrophages (16,44,55). Briefly, fish were anesthetized with MS-222 and bled from the caudal vein to collect autologous serum.…”

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish ( Ictalurus punctatus ) Macrophages

“…Although the pathogenesis of E. ictaluri was reviewed in 1993 (28,31), recent reports demonstrated that E. ictaluri is a facultative intracellular pathogen (3) and that a type III secretion system is required for intracellular survival and replication within channel catfish head kidney-derived macrophages (HKDM) (30). Using signature-tagged mutagenesis (STM) in an immersion challenge model for E. ictaluri, Thune et al (30) identified 50 transconjugants carrying transposon insertions in genes required for survival and replication in the channel catfish host.…”