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Vol. 116: 11–21, 2015 DISEASES OF AQUATIC ORGANISMS Published September 17 doi: 10.3354/dao02902 Dis Aquat Org Dactylogyrus intermedius parasitism enhances Flavobacterium columnare invasion and alters immune-related gene expression in Carassius auratus Chao Zhang1, Dong-liang Li, Cheng Chi, Fei Ling, Gao-xue Wang* Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, PR China 1Present address: Laboratory of Aquatic Ecology, Evolution and Conservation, Department of Biology, KU Leuven, Leuven, Belgium ABSTRACT: The monogenean Dactylogyrus intermedius and the bacterium Flavobacterium columnare are 2 common pathogens in aquaculture. The objective of the present study was to examine the effect of prior parasitism by D. intermedius on the susceptibility of goldfish to F. columnare and to explore the potential immune mechanisms related to the parasite infection. A F. columnare challenge trial was conducted between D. intermedius-parasitized and non- parasitized goldfish. The F. columnare load in gill, kidney, spleen and liver were compared. The expression of immune-related genes (IL-1β2, TNF-α1, TGF-β, iNOS-a, C3 and Lyz) in gill and kidney of D. intermedius-only infected and uninfected control fish were evaluated. D. inter- medius-parasitized goldfish exhibited higher mortality and significantly higher loads (3051 to 537379 genome equivalents [GEs] mg−1) of F. columnare, which were 1.13 to 50.82-fold higher than non-parasitized fish (389 to 17829 GEs mg−1). Furthermore, the immune genes IL-1β2, TNF- α1, iNOS-a and Lyz were up-regulated while the TGF-β and C3 were down-regulated in the gill and kidney of parasite-infected fish compared to the non-parasitized controls. The down-regula- tion TGF-β and C3 was especially noteworthy, as this might indicate the suppression of the host immune functions due to the parasitism by D. intermedius. Taken together, these data demon- strate that parasite infection can enhance bacterial invasion and presents a hypothesis, based on gene expression data, that modulation of host immune response could play a role. KEY WORDS: Bacterial load · RT-PCR · Monogenean · Columnaris · Tumor necrosis factor · Interleukin · Goldfish · Cyprinid Resale or republication not permitted without written consent of the publisher INTRODUCTION tion, as secondary or opportunistic infections may lead to serious disease with adverse consequences In aquaculture systems, host–pathogen interac- such as delay in diagnosis and treatment, which im - tions are rarely one-on-one due to the incredible pedes host recovery (Griffiths et al. 2011, Eswarappa prevalence and diversity of pathogens (Shoemaker et et al. 2012). al. 2008), and individual hosts are often co-infected Over the past few years there has been increasing with multiple disease agents (Xu et al. 2012b). How- evidence that multi-species co-infections contribute ever, most studies on host–pathogen interactions to the severity of some infectious diseases, especially concentrate on a single infection. Studying multi- bacterial diseases (Busch et al. 2003). For example, species infections is gaining more and more atten- Bandilla et al. (2006) demonstrated Argulus coregoni *Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 12 Dis Aquat Org 116: 11–21, 2015 infection could increase the susceptibility of rainbow columnare in different tissues (gill, kidney, spleen trout to Flavobacterium columnare. Recently, it was and liver) of goldfish; and (2) examine the expression shown that parasitism by Ichthyophthirius multifiliis of selected immune genes in gill and kidney of D. enhanced the host’s susceptibility to the bacterium intermedius-infected fish to explore, at the gene Edwardsiella ictaluri and induced more severe mor- level, whether the host’s immune response to D. inter - tality of channel catfish (Xu et al. 2012c). The suscep- medius might contribute to F. columnare suscep - tibility of channel catfish to Aeromonas hydrophila tibility. infection and resulting mortality also increased if the hosts were previously infected with I. multifiliis (Xu et al. 2012a). MATERIALS AND METHODS Although some studies have shown that parasitic infection can enhance bacterial invasion, the precise Fish and parasite samples mechanisms of this phenomenon remain unknown. The possible pathways for such increased suscepti- Four hundred healthy goldfish (mean weight 10.57 bility might be direct. For example, the parasites ± 1.83 g and mean length 7.85 ± 0.26 cm) were ob - might directly damage fish skin and/or gills (Buch- tained from Shaanxi Fisheries Research Institute mann & Bresciani 1997) or the parasite might act as a (Shaanxi, China) and maintained in a glass aquarium vector for a bacterial pathogen (Cusack & Cone 1986, with 200 l aerated tap water (25.0 ± 0.2°C). Twenty Xu et al. 2012b). Additionally, Xu et al. (2012a) spec- goldfish were checked randomly to verify pathogen- ulated that some parasites could enhance the bac - free status of parasites (microscopy) and bacteria (plate terial infection indirectly via reducing the host’s methods and PCR). For this step we only fo cused on immune protection. However, only a few studies some frequently reported pathogenic bacteria: Aero - have investigated this aspect using immuno-physio- monas hydrophila (Wang et al. 2003), Staphylococcus logical approaches, and up until now no studies on aureus (Oliveira & de Lencastre 2002), Vibrio harveyi immune gene regulation have been done (Bowers et (Pang et al. 2006), V. parahaemolyticus (Yung et al. al. 2000, Jørgensen & Buchmann 2007). 1999) and the target Flavobacterium columnare The monogenean Dactylogyrus intermedius and (Panangala et al. 2007). Fish were acclimatized under the bacterium F. columnare are 2 common pathogens laboratory conditions for 7 d. of goldfish Carassius auratus, and they can induce The goldfish infected with Dactylogyrus inter- high mortality and heavy economic losses (Dove & medius were prepared following the methods de - Ernst 1998, O’Halloran 2000). D. intermedius is a scribed in our previous study (Wang et al. 2008). common parasite infecting the gills of cyprinid fishes Briefly, healthy fish were cohabitated with those and causes a serious problem in fish culture (Buch- infected with D. intermedius for 3 wk, and thereafter, mann et al. 1993, 1995). This parasite has 2 to 4 eye- 10 fish were randomly killed and checked for the spots and 1 pair of large anchor hooks; it lives a direct intensity of parasites under microscope. lifecycle without an intermediate host ( ehulková & Gelnar 2006). Heavily infected fish exhibit swollen and pale gills, excessive mucus production and Bacterial isolation and cultivation accelerated respiration, which results in severe dam- age to the host (Wang et al. 2009). F. columnare An isolate of F. columnare (FC-G1) was obtained belongs to the family Flavobacteriaceae and is the from the gills of diseased goldfish and cultured on aetiological agent of columnaris disease. Columnaris Shieh agar plates (Shieh 1980, Decostere et al. 1997) disease shows erosion and necrosis of gills and skin at 27°C. The Shieh medium was composed as fol- of fish and, moreover, leads to osmotic and elec- lows: peptone, 5 mg ml−1; yeast extract, 0.5 mg ml−1; −1 −1 trolyte imbalances and eventually to the death of the sodium acetate, 10 µg ml ; BaCl2 ·(H2O)2, 10 µg ml ; −1 −1 infected fish (Bader et al. 2003, Tripathi et al. 2005). K2HPO4, 100 µg ml ; KH2PO4, 50 µg ml ; MgSO4 · −1 −1 To our knowledge, there is currently no informa- (H2O)7, 300 µg ml ; CaCl2 · (H2O)2, 6.7 µg ml ; −1 −1 tion available on the effect of prior D. intermedius FeSO4 · (H2O)7, 1 µg ml ; NaHCO3, 50 µg ml ; Noble parasitism on infection of the bacterium F. columnare agar, 10 mg ml−1; distilled water, 1000 ml. This isolate in goldfish or whether the host immunocompetence of F. columnare was identified on the basis of colony is involved. Hence, the objectives of this study are to: morphology, Gram stain, standard biochemical tests (1) determine whether prior parasitism by D. inter- (Shewan et al. 1960) and 16S rDNA sequence align- medius would increase the infection load of F. ment (Weisburg et al. 1991). Single colonies were Zhang et al: Pathogen-induced gene expression in goldfish 13 picked from an agar plate and transferred to Shieh collected to examine the immune genes expression. medium, cultured at 27°C under constant shaking for Six random uninfected fish (not exposed to D. inter- 96 h and used to challenge fish. The concentration medius or F. columnare) were used as a control group (colony-forming units per milliliter, CFU ml−1) of F. (Day 0). All tissues were sampled using aseptic tech- columnare was determined through serial 1:10 dilu- nique after fish were anesthetized with an overdose tions using standard plate counts. of MS-222 (Geruien). Tissues for bacterial quantifica- tion were directly stored at −80°C while tissues for gene expressions were first snap frozen in liquid Water quality nitrogen and subsequently put in TRIzol reagent (Invitrogen) stored at −80°C. During trials, the water temperature, pH, salinity, and dissolved oxygen (DO) in tanks were detected with a thermometer, digital pH meter, refractometer, Preparation of genomic DNA from bacteria and portable oxygen meter, respectively. The actual and fish tissues concentrations of ammonia-N and nitrite-N in test solutions were determined using a portable ammonia The genomic DNA (gDNA) of F. columnare (FC- analyzer (Sensidyne). G1) and other bacteria, used for generating a cali - The mean ± SEM of temperature was 25.0 ± 0.2°C, bration curve to determine F. columnare levels in pH was 6.9 ± 0.4, salinity was 20 ± 1 ppt, and OD was infected tissue, and/or to test specificity of F. colum- 6.9 ± 0.9 mg l–1. Concentrations of ammonia-N and nare QPCR assay, were extracted and purified using nitrite-N levels were 0.73 ± 0.21 mg l−1 and 0.11 ± the DNeasy Tissue kit (Qiagen) following the manu- 0.02 mg l−1, respectively.