Journal of Aquatic Animal Health 27:209–216, 2015 C American Fisheries Society 2015 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2015.1094150 ARTICLE Effect of Nanophyetus salmincola and Bacterial Co-Infection on Mortality of Juvenile Chinook Salmon Sean R. Roon and Julie D. Alexander Department of Microbiology, Oregon State University, 226 Nash Hall, Corvallis, Oregon 97331, USA Kym C. Jacobson National Oceanographic and Atmospheric Administration–Fisheries, Northwest Fisheries Science Center, Newport Research Station, 2030 Southeast OSU Drive, Newport, Oregon 97365, USA Jerri L. Bartholomew* Department of Microbiology, Oregon State University, 226 Nash Hall, Corvallis, Oregon 97331, USA Abstract The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon On- corhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environ- ment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmin- cola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations. Downloaded by [Oregon State University] at 08:36 29 March 2016 In a natural system, fish population dynamics can be reg- infection (Graham 2008; Tort 2011). Experimental stud- ulated or limited by microparasites (viruses, bacteria, and ies have shown that co-infection by various macro- and protozoans) and such macroparasites as helminths and arthro- microparasites resulted in increased mortality of fingerling pods, etc. (Lester 1984; Sindermann 1987; Hershberger et al. Channel Catfish Ictalurus punctatus (Labrie et al. 2004; Shoe- 2006; Fujiwara et al. 2011). Effects of “co-infection” by micro- maker et al. 2012; Xu et al. 2012) and higher bacterial loads in and macroparasites on host mortality, recovery, and para- juvenile European Grayling Thymallus thymallus (Pylkkoetal.¨ site transmission rates can have far-reaching effects on dis- 2006). In addition to these synergistic effects, macroparasites ease dynamics in wild populations (Ezenwa and Jolles 2011), can cause direct damage that facilitates secondary infection. For as parasites can interact within a single host (Pedersen and example, mechanical damage from attachment of the ectopara- Fenton 2007). For example, chronic stress and suppressed site Argulus coregoni increased mortality from bacterial infec- host-immune function caused by an initial macroparasite infec- tion in Rainbow Trout Oncorhynchus mykiss (Bandilla et al. tion can increase host susceptibility to a secondary microparasite 2006). *Corresponding author: [email protected] Received February 24, 2015; accepted September 7, 2015 209 210 ROON ET AL. Understanding how macro- and microparasite co-infections under conditions experienced by salmonids at a temperature might affect disease in wild salmonids is critical, given that dis- (18◦C) typically recorded in PNW watersheds during late sum- ease might contribute to declining populations (Arkoosh et al. mer or early autumn (Billman et al. 2014), when conditions 2004; Ogut and Reno 2004; Fujiwara et al. 2011), and that (high temperature, low flow) are favorable to epizootics of these fish are commonly infected by multiple macro- or mi- F. columnare or A. salmonicida (Wakabayashi 1991; Austin and croparasites or both (Ferguson et al. 2011b; Van Gaest et al. Austin 2012). We hypothesize that juvenile Chinook Salmon 2011). In the U.S. Pacific Northwest (PNW), juvenile Chinook having high intensities of encysted N. salmincola metacer- O. tshawytscha and Coho O. kisutch salmon are infected at high cariae will have greater mortality when challenged with bacterial prevalence (60–80%) by the digenean trematode Nanophyetus pathogens than will fish that have no encysted N. salmincola. salmincola (Arkoosh et al. 2004; Ferguson et al. 2011b). Many Here, we present results from two bacterial challenges designed stocks of these species are listed as threatened or endangered to investigate the effects of N. salmincola infection on mortality under the Endangered Species Act (NMFS 2011), and how this from two freshwater bacteria, F. columnare and A. salmonicida. parasite might interact with other pathogens to affect their sur- vival is not fully understood. Salmonids become infected in freshwater when N. salmincola cercariae released from the first METHODS intermediate snail host penetrate and encyst as metacercariae Fish hosts.—Approximately 1,000 juvenile spring Chinook (Bennington and Pratt 1960). Metacercariae can encyst in most Salmon (∼1.5 g each) were obtained from Marion Forks fish fish tissues but have a propensity towards the posterior kidney hatchery (Idanha, Oregon) in June 2013. Fish were transported and muscle (Jacobson et al. 2008). The encysted metacercariae in aerated 95-L transport tanks with 11◦C water to the John survive until the fish is consumed by a definitive host, either a L. Fryer Salmon Disease Laboratory (SDL; Corvallis, Oregon), mammal or piscivorous bird (Schlegel et al. 1968). Studies on where they were held in a 379-L circular, aerated, flow-through the pathogenicity of N. salmincola suggest that physical damage tank supplied with pathogen-free well water (13◦C) for 6 weeks from cercarial penetration and migration can cause mortality in prior to initiation of the study. Unless otherwise noted, all exper- salmon fry exposed to a high number (e.g., 200 cercariae) at a iments were conducted in aerated, flow-through tanks supplied time (Baldwin et al. 1967); if exposed over a period of time, how- with 13◦C water flowing at 2 L/min. Fish were fed 1–2% body ever, the cercariae will encyst as metacercariae without lethal weight/d with appropriately sized fish feed (Bio-Oregon, Inc., effects (Millemann and Knapp 1970). Nonetheless, field and Warrenton, Oregon but not during 24 h before and after bac- laboratory studies indicate that encysted N. salmincola metac- terial challenges. Prior to bacterial challenge, gill and kidney ercariae can have negative, sublethal effects on swimming abil- tissues from 40 randomly sampled fish were tested for bacterial ity (Butler and Millemann 1971; Ferguson et al. 2012), growth pathogens and metacercariae as described below. Results for all (Sandell et al. 2015), osmoregulation enzymes (Ferguson et al. fish were negative. All procedures utilizing fish were approved 2012), and immune function and disease resistance of juvenile by the Oregon State University (OSU) Institutional Animal Care salmonids when challenged with the marine bacteria Vibrio an- and Use Committee. guillarum (Jacobson et al. 2003). Source of N. salmincola.—Freshwater Juga spp. snails were Juvenile salmonids can accumulate N. salmincola metac- periodically collected from coastal rivers (Siletz, Alsea rivers), ercariae throughout their time spent in-river, wherever they Willamette River tributaries (Luckiamute, McKenzie, South overlap with the distribution of the freshwater Juga spp. snail Santiam rivers), and the Willamette River main stem near the host (Furnish 1990). Thus, if N. salmincola metacercariae in- SDL from June to September 2013. Collected snails were trans- Downloaded by [Oregon State University] at 08:36 29 March 2016 fection increases susceptibility to freshwater bacterial infec- ported to the SDL and placed in single wells of a 12-well tissue tions, juvenile salmonids with a high intensity of infection with culture plate (1 snail per well). Wells were filled with 13◦Cwell N. salmincola (number of metacercariae per infected fish) could water and a piece of organic lettuce and were incubated in the be more likely to succumb to disease during their migra- dark at room temperature (21◦C) overnight. The next day, wells tion to the ocean. The bacteria Flavobacterium columnare and were examined for N. salmincola cercariae, the stage infective to Aeromonas salmonicida ssp. salmonicida are ubiquitous in the fish, using a binocular dissection microscope at 50 × magnifica- freshwater environment (Austin and Austin 2012), are present in tion. Any snail shedding the distinctive N. salmincola cercariae subclinical carrier fish or environmental reservoirs (Bullock and was moved to a 100-L flow-through tank at 13◦C where they Stuckey 1975; Suomalainen et al. 2005a; Kunttu et al. 2012), were maintained for up to 8 weeks prior to fish exposure. and have been linked to mortality in hatchery and wild salmonid Exposures to cercariae were conducted in 100-L tanks, in populations in the PNW (Becker and Fujihara 1978; Ogut