WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2013 29 Although the term “stealth pathogen,” as it pertains to bacteria, is normally reserved for pathogens such as Mycoplasma pneumoniae (pneumonia), Borrelia burgdorferi (Lyme disease) and Bartonella henselae (cat scratch disease) in humans and other mammalian hosts, it also can also be used to describe any pathogen that possesses virulence mechanisms that allow it to invade the host and hide within cells, where it cannot easily be recognized by the immune system and cause persistent and chronic infection. They “fly under the radar” of the immune system so to speak. Such is the case with Francisella infections in fish. The tiny Gram negative coccobacillus has virulence mechanisms that allow it to invade a fish host, infect their blood cells (macrophages) that normally function to phagocytize and kill bacterial pathogens, and replicate within macrophages until they ultimately cause them to die through a process known as apoptosis. As the infection spreads through the body of the fish, the disease affects many vital organs, such as the spleen and kidney, ultimately causing death. In this manner, depending on the immune status of the host, francisellosis in fish can manifest as either an acute infection with high mortalities or a chronic infection that can persist for many months. The Genus Francisella The genus Francisella is a member of the class Gammaproteobacteria, order Thiotrichales, and family Francisellaceae. Bacteria of the genus Francisella are characterized as tiny Gramnegative coccobacilli (0.5 × 1.0 µm) that are strictly aerobic, nonmotile, and are catalase positive and oxidase negative. They have a small genome size (1.8-1.9 Mbp) relative to common enteric bacteria such as Escherichia coli (4.6 Mbp). They have been isolated from a wide variety of animals, including more than 200 mammals (including humans), as well as birds, reptiles, crustaceans, mollusks and ticks. They may exist as endosymbionts of insects and ticks that can serve as vectors for disease transmission. Members of the genus have also been found in soil and water samples and as endosymbionts of protists (Forsman et al. 1994, Abd et al. 2003, Scoles 2004, Barns et al. 2005, Keim et al. 2007, Schrallhammer et al. 2010). There are currently four recognized species in the genus: F. tularensis, F. philomiragia, F. noatunensis and F. hispaniensis. The type species F. tularensis, causes the human disease tularemia. Terrestrial and aquatic mammals such as ground Francisella, the “Stealth Pathogen” of Fish John P. Hawke and Esteban Soto squirrels, rabbits, and water rats serve as tularemia reservioirs for transmission via vectors such as ticks, flies and mosquitos. Because of its highly infectious nature in humans, when introduced via the respiratory route, Francisella tularensis has long been considered a potential biological weapon (Johannson et al. 2004). The other mammalian pathogens in the genus are F. philomiragia and F. hispaniensis whereas F. noatunensis is primarily a pathogen of fish. A newly described Francisella sp. has been detected as a pathogen of mollusks (giant abalone) but this strain has not been placed in a taxonomic category (Kamaishi et al. 2010). The fish pathogen F. noatunensis does not survive and grow above 30° C so it is not infectious to humans or other warm-blooded animals. Francisellosis, an Emerging Disease in Coldwater and Warmwater Fish Species Several emerging bacterial diseases of fish have been described in the past 12 years in a wide variety of farmed and wild fish and shellfish, causing significant mortalities and economic losses. Included among the most pathogenic are bacterial infections caused by the rickettsia-like organisms (RLO) including Piscirickettsia salmonis in coldwater species such as Atlantic salmon (Mauel and Miller 2002, Fryer and Hedrick 2003), an unidentified RLO in European seabass (Athanassopoulou et al. 2004), and Francisella spp. in both coldwater and warmwater species (Hsieh 2006, Nylund et al. 2006). Early efforts to isolate and characterize the causative agents of the diseases were hampered by the inability to culture the causative organisms. In fact, they were all initially placed into the category of RLO or piscirickettsia-like organisms (PLO) because the organisms were non-culturable on standard bacteriological agar media and cell culture was necessary to isolate them. Francisella spp. must be cultured on specialized media such as cysteine heart agar supplemented with hemoglobin as an iron source (Fig. 1). It is unclear in many cases if the increase in incidence is the result of spread of the pathogen to new hosts and geographic locations or as a result of the increased technology available to detect them. In many cases amplification of the pathogen DNA by PCR and sequencing of PCR products was used to identify the pathogen before it was actually cultured (Mauel et al. 2007, Birkbeck et al. 2007). (CONTINUED ON PAGE 28) FIGURE 1. Colonies of Francisella noatunensis subsp. orientalis on cysteine heart agar with hemoglobin CHAH. (Photo: John Hawke)
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