https://cpw.cvlcollections.org/files/original/7ff03b98ba599a39e879fd6b19df3c66.pdf 0d0cd001381bb98f48297b6bedbd3433 PDF Text Text The research in this publication was partially or fully funded by Colorado Parks and Wildlife. Dan Prenzlow, Director, Colorado Parks and Wildlife • Parks and Wildlife Commission: Marvin McDaniel, Chair • Carrie Besnette Hauser, Vice-Chair Marie Haskett, Secretary • Taishya Adams • Betsy Blecha • Charles Garcia • Dallas May • Duke Phillips, IV • Luke B. Schafer • James Jay Tutchton • Eden Vardy �Received: 13 December 2021 | Revised: 17 February 2022 | Accepted: 22 February 2022 DOI: 10.1111/jfd.13605 RESEARCH ARTICLE Dual resistance to Flavobacterium psychrophilum and Myxobolus cerebralis in rainbow trout (Oncorhynchus mykiss, Walbaum) Brian W. Avila1 | Dana L. Winkelman2 | Eric R. Fetherman3 1 Colorado Cooperative Fish and Wildlife Research Unit, Colorado State University, Fort Collins, Colorado, USA 2 U.S. Geological Survey, Colorado Cooperative Fish and Wildlife Research Unit, Department of Fish, Wildlife and Conservation Biology, Colorado State University, Fort Collins, Colorado, USA 3 Colorado Parks and Wildlife, Fort Collins, Colorado, USA Correspondence Brian W. Avila, Colorado Cooperative Fish and Wildlife Research Unit, Colorado State University, Fort Collins, Colorado 80523, USA. Email: bavila@rams.colostate.edu Funding information Federal Aid in Sport Fish Restoration Program, project F-­394, and by the Colorado Parks and Wildlife Aquatic Research Section, Sport Fish Research Studies Abstract Aquatic pathogens are a major concern for fish hatchery production, fisheries management, and conservation, and disease control needs to be addressed. Two important salmonid pathogens are Myxobolus cerebralis and Flavobacterium psychrophilum that cause whirling disease and bacterial coldwater disease (BCWD), respectively. Innate disease resistance is a potential option for reducing disease-­related mortality in hatchery-­reared rainbow trout (Oncorhynchus mykiss, Walbaum). Two experiments were conducted to assess pathogen resistance of first-­generation (F1) rainbow trout created by crossing M. cerebralis-­ and F. psychrophilum-­resistant strains. In the first experiment, we exposed two rainbow trout strains and one F1 cross to six treatments: control (no exposure), mock injection, F. psychrophilum only, M. cerebralis only, F. psychrophilum then M. cerebralis, and M. cerebralis then F. psychrophilum. Results indicated that the F1 cross was not resistant to either pathogen. In the second experiment, we exposed five rainbow trout strains and four rainbow trout crosses to F. psychrophilum. The second experiment indicated that at least one rainbow trout cross was F. psychrophilum-­resistant. Achieving dual resistance may be possible using selective breeding but only some multigenerational strains are suitable candidates for further evaluation. KEYWORDS bacterial coldwater disease, Flavobacterium psychrophilum, Myxobolus cerebralis, rainbow trout, whirling disease 1 | I NTRO D U C TI O N losses to producers of salmon and rainbow trout (Oncorhynchus mykiss, Walbaum) (Antaya, 2008). As a result, BCWD is considered Flavobacterium psychrophilum, the causative agent of bacterial one of the most important hatchery diseases in the world (Michel coldwater disease (BCWD), is found in cultured and wild fishes et al., 1999). Infections typically affect age-­0 salmonids (Cipriano & worldwide and causes significant infection in captive salmonid pop- Holt, 2005; Nicolas et al., 2008) but can also affect larger and older ulations (LaFrentz & Cain, 2004; Starliper, 2011). Mortality associ- fish (LaFrentz & Cain, 2004). Infected fish show a broad range of ated with infections can be as high as 90% (Barnes & Brown, 2011; clinical disease signs such as discoloration of the adipose fin, lesions, Nilsen et al., 2011) depending on water temperature and devel- spiral swimming behaviour, “blacktail”, spinal deformities, and pale opmental stage of the host (Decostere et al., 2001; Wood, 1974). or necrotic gills (Borg, 1948; Davis, 1946; Kent et al., 1989; Martinez Outbreaks causing high mortality can result in massive economic et al., 2004; Ostland et al., 1997). This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd. J Fish Dis. 2022;00:1–13. � wileyonlinelibrary.com/journal/jfd | 1 �2 | AVILA et al. Antibiotics are the most used treatment for an F. psychroph- trout to re-­ establish populations in the presence of the parasite ilum infection. Oxytetracycline (OTC) has been used worldwide (Avila et al., 2018; Fetherman et al., 2014), and reproduction and (Branson, 1998; Groff & LaPatra, 2001; LaFrentz & Cain, 2004; recruitment are occurring (Fetherman et al., 2014). Stocking F. Lumsden et al., 2006; Post, 1987), and amoxicillin and oxolinic acid have psychrophilum-­resistant fish with no resistance to M. cerebralis could been used throughout Europe (Branson, 1998; Bruun et al., 2000). result in failure due to mortality associated with M. cerebralis ex- Several studies suggest that antimicrobial resistance is occurring posure, as well as increased infection severity and loss of progress in treated populations (Bruun et al., 2000; Schrag & Wiener, 1995). gained from M. cerebralis-­resistant rainbow trout stocking efforts. Starting in 1986, oxolinic acid was used to treat F. psychrophilum in For F. psychrophilum-­resistant fish to be a viable management tool, Denmark hatcheries, but by 2000, the bacteria were 100% resistant to it is imperative to determine if the PRR exhibit any resistance to M. this treatment. Between 1994 and 1998, 60%–­75% of F. psychrophilum cerebralis, understand if resistance to both F. psychrophilum and M. ce- in Danish hatcheries showed resistance to both OTC and amoxicillin rebralis is compatible and achievable, and, given the possibility for ex- (Bruun et al., 2000). Another potential treatment option for BCWD is posure to either or both pathogens in a hatchery or wild environment, vaccination, and though development of a vaccine has been attempted understand how they might interact when dual exposure occurs. (Sudheesh & Cain, 2016), none are currently commercially available. Our overall goal was to determine if crossing strains of rainbow Due to concerns about antibiotic resistance and the lack of trout resistant to each pathogen would result in progeny that were a vaccine, other strategies to prevent F. psychrophilum infec- genetically resistant to both pathogens, and this was evaluated using tions warrant investigation. Hadidi et al. (2008) suggested using two experiments. The goal of the first experiment was to determine a genetically resistant brood fish to manage BCWD outbreaks. In if it was possible to develop dual resistance by crossing a rainbow 2005, the US Department of Agriculture-­ A gricultural Research trout resistant to M. cerebralis with the PRR. We also wanted to un- Service's (USDA-­ ARS) National Center for Cool and Cold Water derstand the possible effects of coinfection when fish were exposed Aquaculture (NCCWA) developed a program to create a rainbow to both pathogens and if the order of exposure was important. Fish trout strain that was genetically resistant to F. psychrophilum (Hadidi could be infected with F. psychrophilum in the hatchery and then be et al., 2008; Leeds et al., 2010). The strains used to create the F. stocked into the wild and exposed to M. cerebralis. Conversely, in an psychrophilum-­resistant population were chosen based on known M. cerebralis-­positive hatchery, fish may be exposed to M. cerebralis genetic and domestication history including the Ennis National Fish first followed shortly after by F. psychrophilum exposure. The goal Hatchery Shasta strain; College of Southern Idaho, House Creek of our second experiment was to determine if it was possible to de- strain; Kamloops/Puget Sound Steelhead cross; and University of velop a first-­generation (F1) rainbow trout cross that was resistant Washington, Donaldson strain (Silverstein et al., 2009). The result- to F. psychrophilum when crossing pure parental strains resistant to ing F. psychrophilum-­resistant rainbow trout, the ARS-­Fp-­R strain, either F. psychrophilum or M. cerebralis. showed reduced mortality when exposed to F. psychrophilum (Leeds et al., 2010; Wiens et al., 2013). A third-­generation lot of ARS-­ Fp-­R was sent to Utah Division of Wildlife Resources (UDWR), and then in 2016, Colorado Parks and Wildlife (CPW) imported the F. psychrophilum-­resistant rainbow trout from UDWR to be used in the 2 | M E TH O D S 2.1 | Rainbow trout strains and crosses CPW hatchery system to manage mortality due to F. psychrophilum infections. Within the CPW hatchery system, these imported fish Three strains of M. cerebralis-­resistant rainbow trout were used for are known as psychrophilum-­resistant rainbow (PRR). As of 2020, both experiments, German rainbow trout, Harrison Lake rainbow the USDA-­ ARS NCCWA had produced its fifth generation of F. trout, and the German Rainbow × Harrison Lake rainbow trout psychrophilum-­ resistant rainbow trout (G. Weins, USDA personal (Table 1). The pure German Rainbow (GR) is a domesticated hatch- communication, June 18, 2019). A similar approach has been used ery rainbow trout which was exposed to M. cerebralis over many to produce whirling disease Myxobolus cerebralis resistant rainbow generations in Germany and is more resistant to M. cerebralis than trout and reestablish rainbow trout fisheries in the presence of the many other rainbow trout strains found in North America (Hedrick parasite (Fetherman et al., 2011, 2012, 2014; Schisler et al., 2006). et al., 2003). The Harrison Lake rainbow trout (HL; origin: Harrison Using F. psychrophilum-­resistant fish in hatcheries system may Lake, Montana; Wagner et al., 2006) is one of the wild rainbow trout provide a valuable tool to reduce mortality due to outbreaks of strains that were crossed with the GR to create a fish capable of sur- BCWD, and F. psychrophilum-­resistant rainbow trout, or PRR, were viving and reproducing in the wild (Fetherman et al., 2015; Schisler brought into the Colorado's hatchery system in 2016 for that pur- & Fetherman, 2009). The GR × HL used within Experiment 1 are pose. However, it is unknown whether the PRR are also resistant 87.5% GR and 12.5% HL and has been propagated as a hatchery to M. cerebralis. M. cerebralis was introduced to Colorado in the late brood stock since 2006 (Schisler et al., 2011). Despite showing M. 1980s, and later found in free-­ranging salmonid populations in 11 cerebralis resistance, the GR × HL shows some of the highest mortal- of the state's 15 major river drainages (Barney et al., 1988; Nehring ity in the CPW hatchery system due to F. psychrophilum infections. & Thompson, 2003), resulting in the collapse of wild rainbow trout In both experiments, we used the PRR from Colorado and the fifth-­ populations throughout Colorado (Nehring & Thompson, 2001). generation ARS-­Fp-­R from the USDA-­ARS NCCWA to investigate F. The state of Colorado has been using M. cerebralis-­resistant rainbow psychrophilum resistance (Table 1). �| AVILA et al. 3 TA B L E 1 Rainbow trout strains used for each experiment and their known pathogen resistance Rainbow Trout Strains/Crosses Abbreviation Experiment Resistance Fish type German Rainbow × Harrison Lake GR × HL 1 M. cerebralis Strain (German Rainbow × Harrison Lake) × psychrophilum-­resistant rainbow GHP 1 Unknown F1-­generation cross psychrophilum-­resistant rainbow PRR 1, 2 F. psychrophilum Strain Harrison Lake HL 2 M. cerebralis Strain Germain Rainbow GR 2 M. cerebralis Strain S-­Line ARS-­Fp-­S 2 Unknown Strain Agricultural Research Service -­F. psychrophilum -­Resistant ARS-­Fp-­R 2 F. psychrophilum Strain Harrison Lake × psychrophilum-­resistant rainbow HL × PRR 2 Unknown F1-­generation cross Harrison Lake × Agricultural Research Service -­ F. psychrophilum -­Resistant HL × ARS-­Fp-­R 2 Unknown F1-­generation cross German Rainbow × psychrophilum-­resistant rainbow GR × PRR 2 Unknown F1-­generation cross German Rainbow × Agricultural Research Service -­F. psychrophilum -­Resistant GR × ARS-­Fp-­R 2 Unknown F1-­generation cross F I G U R E 1 Experimental design for Experiment 1 (a) and Experiment 2 (b). Three strains or crosses were used in Experiment 1. Each strain was exposed to six treatments, with the number of tanks for each treatment denoted by [ ] for a total of 108 tanks. Nine strains or crosses were used in Experiment 2 and evaluated only for resistance to F. psychrophilum using two treatments and a total of 84 tanks 2.2 | Experiment 1 –­dual exposure to Flavobacterium psychrophilum and Myxobolus cerebralis pathogens. These strains were spawned at the CPW Crystal River Hatchery (Carbondale, Colorado) in January 2019 and then transported as eyed eggs to the CPW Bellvue Fish Research Hatchery Two strains and one cross of rainbow trout were used for this (Bellvue, Colorado) for hatching. Fish were moved from the CPW dual exposure experiment (Table 1; Figure 1), the PRR, which is Bellvue Fish Research Hatchery, at eight weeks post-­hatch to a resistant to F. psychrophilum, the GR × HL, which is resistant to laboratory located on the Colorado State University (CSU) main M. cerebralis, and the cross of the GR × HL and PRR (GHP), which campus. Fish were moved two days prior to the beginning of the was created and evaluated for maintaining resistance to both experiment. �4 | AVILA et al. The PRR, GR × HL, and GHP were exposed to six treatments: psychrophilum only, F. psychrophilum and then M. cerebralis) and M. (1) no pathogen exposure (control), (2) mock injection, (3) F. cerebralis treatments (M. cerebralis only, M. cerebralis and then F. psy- psychrophilum-­only exposure, (4) M. cerebralis-­only exposure, (5) F. chrophilum). The same exposure methods described above were used psychrophilum exposure followed by M. cerebralis exposure four days for the dual exposures, but some (n = 18 tanks each) were exposed to later, and (6) M. cerebralis exposure followed by F. psychrophilum ex- F. psychrophilum and then M. cerebralis four days later or exposed to posure four days later. Each strain and treatment combination had six M. cerebralis and then F. psychrophilum four days later. replicates, resulting in 108 total twenty-­gallon (76-­L) tanks. Thirty-­ Experiment 1 had two objectives. The first was to conduct five individual rainbow trout of the assigned strain/cross were con- an F. psychrophilum exposure experiment to observe mortality. tained in each tank, resulting in 3780 fish in the experiment. Water Flavobacterium psychrophilum exposures were conducted on day (13.4ºC ± 2.1 SD) was sourced from the city and dechlorinated by zero and day four and fish were held for 28 days so that mortality running through large, activated charcoal filters. Tanks were set up could stabilize (showing no more mortality) because fish can survive for flow-­through water exchange at a flow of 15 gallons per hour. and recover from F. psychrophilum infections. The second objec- To limit potential cross-­contamination from pathogen-­exposed tive started after the completion of the F. psychrophilum portion of tanks, control tanks were located on the top shelf of the three-­tier the experiment. All remaining fish were reared until they reached shelving system. Strains were randomly assigned to tanks and either 2347.8 ± 73.3 SD degree-­days, which was necessary to ensure full a control or mock injection treatment within the top shelf of the sys- development of myxospores in the treatments with M. cerebralis. tem. Pathogen exposure treatments and strains were then randomly Tanks were cleaned every two weeks on a rotating schedule. assigned to the remaining tanks on the top shelf (note that control/ Throughout the rearing process all tanks were monitored twice daily mock injection tanks were never located next to pathogen exposure and moribund and dead fish in each tank were measured, weighed, tanks on the same shelving unit) and the tanks on the other two tiers signs of disease were documented, and the fish were then removed. of the shelving system. Fish were fed twice a day at the standardized feeding rate (per Flavobacterium psychrophilum culture and preparation are described cent body weight per day [% BW/d]) based on the manufacturer's in Avila (2021). An initial batch weight of the fish in each tank was taken (BioOregon) suggested specifications of fish size and rearing tem- and used to calculate the amount of feed per day (g) and the dose of perature. Feed amount was adjusted daily based on the number of F. psychrophilum given the average weight per fish (PRR: 0.46 g ± 0.03 fish within each tank. Batch weights used to adjust feeding rates SD; GR × HL: 0.41 g ± 0.05; GHP: 0.48 g ± 0.06). For F. psychrophilum were taken from each tank by placing all fish from the tank into a exposure, rainbow trout were first sedated using MS-­222 (90 mg/ml of tared water bucket on a scale, obtaining individual weights by di- water) and then injected subcutaneously at the dorsal midline poste- viding the total weight by the known number of fish, and calculating rior to the dorsal fin with 8.8 × 106 colony forming units per millilitre the grams per fish. Batch weighing was conducted every two weeks (CFU/ml; 25 μl) of virulent F. psychrophilum (CSF259-­93 obtained from starting 28 days post-­exposure to prevent affecting mortality results K. Cain, Moscow, Idaho). For the mock injection, rainbow trout were in the classic F. psychrophilum exposure experiment by handling fish. similarly subcutaneously injected with 25 μl of tryptone yeast extracts After reaching the required degree days for myxospore devel- and salt (TYES) to verify that exposure to the bacteria and not physical opment, all surviving fish were euthanized, weighed, measured, injury from injection caused mortality. No injections occurred for fish and inspected for clinical signs of whirling disease and/or BCWD. in the control, M. cerebralis only, or, initially, the M. cerebralis exposure Euthanized fish had their heads removed from the body just behind followed by F. psychrophilum treatments. the operculum and pectoral fins and placed in individually labelled Myxobolus cerebralis triactinomyxons (TAMs), the waterborne bags and frozen (Fetherman et al., 2012). Myxospores were enumer- infectious stage of the parasite, were produced by Tubifex tubifex ated (O’Grodnick, 1975) using pepsin–­trypsin digest (PTD; Markiw worm cultures maintained at the CPW Parvin Lake Research Station & Wolf, 1974a, 1974b). The processing of fish was initiated at the (Red Feather Lakes, Colorado). The concentration of viable TAMs CPW Aquatic Animal Health Laboratory (AAHL; Brush, Colorado) was estimated by mixing 1000 μl of filtrate containing TAMs and and conducted by the AAHL staff. A subset of samples were pro- 60 μl of crystal violet; 84.6 μl of this mixture was then placed on cessed entirely by the AAHL, including pepsin-­trypsin digestion and a slide and the number of TAMs per slide was counted. Ten TAM myxospore counting. The remaining fish were digested using pepsin counts were conducted out of the filtrate to get an average num- at the AAHL and then transferred to the Colorado Cooperative Fish ber of TAMs per mL, and fish (732.2 ± 34.6 degree-­days [°C*days] and Wildlife Research Unit laboratory to finish trypsin digestion and post-­hatch) were exposed to 2,000 TAMs per individual for a total myxospore counting. The same methods were used in both labora- of 70,000 TAMs per tank (following Fetherman et al., 2011). Fish in tories to ensure consistency in the results. the control treatment, mock injection, F. psychrophilum only, and, initially, F. psychrophilum exposure followed by M. cerebralis exposure were not exposed to TAMs. 2.2.1 | Statistical analysis On the first day of the experiment (day zero), mock injections were conducted first to prevent accidental exposures to F. psychrophilum The statistical analysis focused on five endpoints: (1) 28-­day post-­ using the same injectors, followed by F. psychrophilum treatments (F. exposure mortality, (2) end of experiment mortality, (3) differences in �| AVILA et al. 5 growth, (4) disease signs, and (5) myxospore counts for M. cerebralis-­ was evidence of differences in myxospore counts, then a pairwise exposed tanks. comparison with a Tukey adjustment was used to compare among strains and treatments. 2.2.2 | Mortality 2.2.5 | Clinical signs of whirling disease Cumulative per cent mortality (CPM), the number of dead fish divided by the total number of fish at the start, was calculated for each tank Clinical whirling disease signs included cranial deformities, spinal de- for the first 28 days post-­exposure and at the end of the experiment. formities, opercular deformities, exophthalmia, lower jaw deformi- A chi-­squared test was used to determine if there were differences ties, and blacktail. We observed bubbles on the fins and near the gills in mortality (a proportion) among the strains, treatments, and strain on some individuals, indicating the potential for gas bubble disease by treatment interaction (strain*treatment). If there was evidence of at the end of the experiment. No additional effects of mortality were a difference in mortality, pairwise comparisons with a Tukey adjust- observed due to gas bubble disease; however, exophthalmia was re- ment were used to compare among strains and treatments. moved from the analysis because it might have been due to gas bubble disease and not exposure to M. cererbralis. A logistic regression was used to fit the data with the proportion of individuals in a tank 2.2.3 | Growth as the response and strain, treatment, and the interaction between strain and treatment as the factors that predict clinical signs of dis- The difference in weight at 28 days post-­exposure was analysed ease. If there was evidence of a difference in clinical signs of disease, using ANOVA with strain, treatment, and strain*treatment as the then a pairwise comparison with a Tukey adjustment was used to factors to explain differences in growth due to exposure to F. psy- compare among strains and treatments. chrophilum. The difference in weight at the end of the experiment was analysed using an ANCOVA with strain, treatment, and interaction between strain and treatment as the factors, and the number of 2.3 | Experiment 2 fish within a tank at the end of the experiment as a covariate as tank density was thought to explain differences in growth. The differ- F1-­ generation crosses were created by crossing pure German ence in weight at the end of the experiment may also show if growth Rainbow (GR) and pure Harrison Lake rainbow trout (HL) with ARS-­ was affected by surviving F. psychrophilum infection because tradi- Fp-­R obtained from the USDA-­ARS NCCWA or PRR obtained from tional F. psychrophilum exposure experiments are only conducted for CPW. All strains were created in collaboration with the USDA-­ARS 28 days. If there was evidence of a difference in growth in either NCCWA, the CPW Crystal River Hatchery, and the CPW Bellvue model, then a pairwise comparison with a Tukey adjustment was im- Fish Research Hatchery. The NCCWA provided ARS-­Fp-­R milt and plemented. Type III sums of squares were used to account for the the Crystal River Hatchery provided PRR milt, and milt from these unbalanced number of tanks and statistical significance was inferred sources were crossed with pure GR and pure HL eggs at the Bellvue at the α = 0.05 level. Fish Research Hatchery in January 2020. All F1 crosses were made using F. psychrophilum-­ resistant males and M. cerebralis-­resistant 2.2.4 | Myxospore counts females. The spawning resulted in first-­ generation HL × PRR, HL × ARS-­Fp-­R , GR × PRR, and GR × ARS-­Fp-­R crosses (Table 1; Figure 1). The Crystal River Hatchery produced pure PRR rain- To account for myxospore counts of zero in the control, mock in- bow trout, whereas the NCCWA produced the ARS-­Fp-­R and an jection, and F. psychrophilum-­only exposure, a two-­part modelling F. psychrophilum-­susceptible line rainbow trout (S-­Line), and both approach was taken. First, we used a logistic regression to quan- facilities shipped eyed eggs to the Bellvue Fish Research Hatchery tify the difference in myxospore counts between the tanks not ex- where they were hatched. posed to M. cerebralis to those that were exposed to M. cerebralis. Fish were moved from the Bellevue Fish Research Hatchery to a The response is specified by a binary variable (0 if not exposed to laboratory located on the CSU main campus. An initial sample weight M. cerebralis or 1 if a fish was exposed to M. cerebralis) with the pre- of each tank was taken prior to moving fish to obtain average indi- dictor variables of strain, treatment, and the strain by treatment in- vidual fish weights for each strain or cross (HL: 0.78 g ± 0.06 (SD); teraction. Chi-­squared values were then used to determine if there HL × PRR: 1 g ± 0; HL × ARS-­Fp-­R: 1 g ± 0; GR: 1 g ± 0; GR × PRR: were statistical differences in the number of myxospores between 1.18 g ± 0.06; GR × ARS-­Fp-­R: 1.24 g ± 0.09; PRR: 1.46 g ± 0.10; the predictor variables. Second, we used a negative-­binomial regres- S-­Line: 1.1 g ± 0.14; ARS-­Fp-­R: 1.1 g ± 0.12). Weights were used to sion, which helped account for overdispersion of the data (Boulton calculate the total amount of feed per day (g) for each tank and F. & Williford, 2018; Duan et al., 1983), to compare the average dif- psychrophilum dosage. Control fish were randomly assigned to tanks ference in myxospore count among strains, treatments, and their located on the top shelf of the three-­tier shelving system to limit po- interaction in only fish that were exposed to M. cerebralis. If there tential bacterial contamination of control tanks, and F. psychrophilum �6 | AVILA et al. treatment tanks were randomly assigned to the remaining tanks on exposure treatments compared to controls, TYES, and M. cerebra- the lower two tiers. Each twenty-­gallon (76-­L) tank held twenty-­ lis treatments. Mortality did not differ between the GR × HL and five individual rainbow trout of the assigned strain/cross. Water GHP when exposed to F. psychrophilum. However, mortality for (10°C ± 0.77 SD) was sourced from the city and dechlorinated by the PRR was lower than either the GR × HL or GHP when exposed running through large, activated charcoal filters. Fish tanks were set to F. psychrophilum. up for flow-­through water exchange at a flow of 15 gallons per hour. Mean mortality at the end of the experiment ranged from 9% to We compared five pure strains of rainbow trout, GR, HL, ARS-­ 100% (Figure 2). The chi-­squared test indicated an interaction be- Fp-­R , PRR and the S-­Line, and four F1-­generation crosses, GR × ARS-­ tween strain and treatment (χ2 = 428.12, p-­value <.01). The control Fp-­R , HL × ARS-­Fp-­R , GR × PRR and HL × PRR. All nine were injected and TYES treatments showed low mortality and did not differ among subcutaneously posterior to the dorsal fin above the midline with strains (Figure 2a). Flavobacterium psychrophilum-­only exposure caused virulent 8.8x106 CFU/mL of F. psychrophilum (CSF259-­93; 25 μl), high mortality in GR × HL and GHP but was significantly lower in PRR, with ten replicates (tanks) each except for the GR × ARS-­Fp-­R (five indicating that dual resistance was not achieved (Figure 2b). Myxobolus tanks) which had high mortalities in the hatchery, and the S-­Line cerebralis-­only exposure caused high mortality in PRR but was signifi- rainbow trout (two tanks). Mock injections were given to four strains cantly lower in GRR and GR × HL (Figure 2b), indicating that the PRR (GR, HL, ARS-­Fp-­R and HL × ARS-­Fp-­R), with two replicates each, is susceptible to M. cerebralis. Mortality in GRR and GR × HL exposed and injected similarly with TYES (25 μl). We did not include mock to M. cerebralis was not distinguishable from the control or TYES treat- injections for every strain or equal numbers of replicates because ments (Figure 2a,b). Dual exposures resulted in higher mortality com- water resources were limited in the laboratory. Fish were monitored pared to F. psychrophilum-­only and M. cerebralis-­only exposures for the twice a day after injections. Moribund and dead fish were removed PRR but not the GR × HL or GHP (Figure 2). from each tank and recorded for 28-­days after injection. At the end of the rearing period, all remaining fish were euthanized. 3.1.2 | Myxospore counts 2.3.1 | Statistical analysis As expected, none of the fish in the control, F. psychrophilum-­only, and TYES treatments had myxospores. Mean myxospore counts Cumulative per cent mortality (CPM) was calculated for each tank at in the M. cerebralis exposure treatments (M. cerebralis only, F. psy- the end of the 28-­day experiment. A chi-­squared test was used to de- chrophilum followed by M. cerebralis exposure, and M. cerebralis termine if there was a relationship between CPM and strain, exposure, followed by F. psychrophilum exposure) ranged between 556 (556; and the interaction between strain and exposure (strain*exposure) and SE) and 645,201 (130,651; SE) per fish. The chi-­squared test indi- if there was a relationship between mortality and strain dependent cated that there were differences between treatments exposed to on fish weight (strain*weight). If there was evidence of a difference M. cerebralis and those that were not exposed (χ2 = 106.45, p-­value in mortality, then Tukey adjusted pairwise comparisons were used to <.01). The probability of having a myxospore count greater than zero compare among strains and treatments. Finally, a logistic regression was 99.9% when exposed to M. cerebralis compared to a probability was used to estimate the probability of mortality based on factors that of zero when not exposed to M. cerebralis (logistic regression). The significantly affected CPM identified in the chi-­square analysis. negative-­binomial regression indicated a strain*treatment interaction (χ2 = 22.80, p-­value <.01). There were statistically significant 3 | R E S U LT S 3.1 | Experiment 1 differences among the PRR, GHP, and GR × HL when exposed to M. cerebralis only. The GR × HL strain developed the lowest number of mean myxospores (77,569 ± 9032; SE), followed by GHP (224,553 ± 21,704; SE) and then PRR (645,201 ± 130,651; SE; Figure 3). When fish survived exposure to F. psychrophilum, average 3.1.1 | Mortality myxospore counts were lower in the F. psychrophilum followed by M. cerebralis exposure treatment compared to the M. cerebralis-­only For all three strains, control and TYES treatment CPMs were treatment (Figure 3). For the GHP, exposure to M. cerebralis followed low (0-­-­10%) and did not differ at 28 days post-­exposure. The by exposure to F. psychrophilum resulted in significantly higher myx- single exposures to F. psychrophilum only and M. cerebralis only ospore counts. resulted in CPMs ranging from 32%–­9 0% and 0%–­1% at 28 days post-­exposure, respectively. The CPMs for the dual exposures, F. psychrophilum followed by M. cerebralis exposure and M. cerebralis 3.1.3 | Clinical signs of whirling disease followed by F. psychrophilum exposure, ranged from 46%–­94% and 58%–­99%, respectively. The chi-­s quared test indicated an inter- Clinical signs of whirling disease (cranial deformities, spinal de- action between strain and treatment (χ2 = 70.17, p-­value <.01). formities, opercular deformities, lower jaw deformities, and black- Mortality at 28 days was significantly higher in F. psychrophilum tail) developed between two and three months post-­exposure. �| AVILA et al. 7 F I G U R E 2 Cumulative per cent mortality (CPM) by strain and treatment at the end of Experiment 1. (a) Control and mock injection (TYES), (b) Flavobacterium psychrophilum only (Fp) and Myxobolus cerebralis only (Mc), and (c) F. psychrophilum followed by M. cerebralis (FpMc) and M. cerebralis followed by F. psychrophilum (McFp). Black lines within the boxes indicate the median of the distribution. Box and whisker plots with the same letter indicate no significant differences and box and whisker plots with different letters indicate statistically significant differences Both the GHP cross and PRR strain exhibited classic whirling 3.2 | Experiment 2 swimming behaviour when fish were startled. The PRR strain had the most visible signs of whirling disease, with a significantly Mortalities started within the first two days post-­exposure for the higher proportion of blacktail visible compared to the GR × HL and ARS-­Fp-­R and the S-­Line strains compared to the other strains, which GHP and also showed extreme spinal deformities (Supplementary started between three and five days post-­exposure (Supplementary Materials; S1). Four out of the five clinical signs of disease (cranial, Materials; S2). There were no mortalities within the mock injection spinal, opercular, and lower jaw deformities) showed evidence of a controls. strain by treatment interaction (p-­value <.01), with the PRR devel- Chi-­ s quared tests indicated that fish weight did not have oping a higher percentage of these deformities in the M. cerebra- an effect on mortality among strains (strain*weight; χ2 = 9.68, lis exposure treatments. None of the fish in F. psychrophilum-­only p-­value = .08) and there was not an interaction between strain treatments developed a higher percentage of deformities than control fish. and exposure (χ2 = 0, p-­value = 1). However, there was a difference in mortality by both strain and exposure (χ2strain = 544.95; �8 | AVILA et al. F I G U R E 3 Mean myxospore count per fish by rainbow trout strain and treatment (TYES = mock injection, Fp = Flavobacterium psychrophilum only, Mc = Myxobolus cerebralis only, FpMc = exposed to F. psychrophilum followed by M. cerebralis, and McFp = exposed to M. cerebralis followed by F. psychrophilum) at the end of Experiment 1. No data available for GR × HL McFp treatment because of 100% mortality before the end of the experiment. Black lines within the boxes indicate the median of the distribution. Box and whisker plots with the same letter indicate no significant differences and box and whisker plots with different letters indicate statistically significant differences F I G U R E 4 Cumulative per cent mortality by strain/cross for the fish exposed to F. psychrophilum only (mock injections not included) in Experiment 2. Black lines within the boxes indicate the median of the distribution. Box and whisker plots with the same letter indicate no significant differences and box and whisker plots with different letters indicate statistically significant differences χ2exposure = 493.10; p-­value <.01). The TYES control groups showed not resistant to F. psychrophilum, which was similar to experiment no mortality associated with injection. The estimated probabili- one. The PRR strain had significantly less mortality compared to ties of mortality for the strains in the F. psychrophilum exposure the ARS-­Fp-­R strain (Figure 4), indicating that the PRR is more re- ranged from 19.3%–­98.8%. The S-­L ine, which was used as a pos- sistant to F. psychrophilum than ARS-­Fp-­R . The GR × ARS-­Fp-­R and itive control, showed expected high mortality (Figure 4). The S-­ GR × PRR showed less mortality than the GR strain and similar to Line mortality was not different from that of the HL or GR strains that of the ARS-­Fp-­R (Figure 4) suggesting that some resistance (Figure 4), indicating that the M. cerebralis-­resistant strains were was transferred from the F. psychrophilum-­resistant strains and �| AVILA et al. 9 indicating F. psychrophilum resistance in the F1 generation. The cerebralis resistance in HL × PRR would be required to determine if HL × ARS-­Fp-­R and HL × PRR showed less mortality than the HL the HL × PRR retains M. cerebralis resistance. strain (Figure 4) suggesting resistance was transferred from the The GR × PRR or GR × ARS-­Fp-­R may still be a viable option for F. psychrophilum-­resistant strains. In addition, the HL × ARS-­Fp-­R the development of dual resistance to both M. cerebralis and F. psy- and HL × PRR showed the lowest mortality compared to the GR, chrophilum. The reduction in mortality associated with F. psychroph- HL, GR × ARS-­Fp-­R , and GR × PRR, suggesting that F. psychrophi- ilum exposure was not as large as that seen in the HL × PRR but was lum resistance can be passed on to F1 progeny. The HL × PRR and still significant. Additionally, first-­ generation rainbow trout cross the PRR strains had the lowest mortality compared to the other progeny of the GR and the Colorado River rainbow trout (CRR) have nine, indicating the highest F. psychrophilum resistance among the shown high resistance to M. cerebralis (Fetherman et al., 2012) sug- strains. gesting that both GR × PRR or GR × ARS-­Fp-­R may retain resistance to M. cerebralis. However, resistance to M. cerebralis in the GR × PRR 4 | DISCUSSION and GR × ARS-­Fp-­R should still be confirmed. Developing rainbow trout strains that are resistant to multiple pathogens will require an understanding of the immunological re- The overall objective of our experiments was to evaluate the po- sponses to each pathogen. Our research was not designed to ad- tential of developing rainbow trout that were resistant to both M. dress immunological responses; however, innate and acquired cerebralis and F. psychrophilum, suitable for use in both the hatchery immune responses to each pathogen are undoubtedly complex system and for stocking in aquatic systems in which M. cerebralis is (Cox, 2001). In dual infections such as ours, the effect of both in- established. We investigated the consequences of infection with fectious agents could be increased, suppressed, or one may be in- each pathogen and coinfection with both pathogens on two rainbow creased and the other suppressed, and the ultimate result may be trout strains and one cross, and F. psychrophilum exposure effects in hard to predict (Cox, 2001). Specific genes and innate resistance pure strains and F1-­generation crosses. It appears that some crosses have been reported for rainbow trout immune response to M. ce- might be useful in the development of rainbow trout that are resist- rebralis (Baerwald et al., 2008; Saleh et al., 2019). Exposure to M. ant to both pathogens. However, others do not appear to have that cerebralis results in the activation of the cytokine gene IL-­1β that potential. Strains known for their resistance to M. cerebralis were not is a part of the innate immune system in fishes (Baerwald, 2013). resistant to F. psychrophilum, and vice versa, strains known for their The IL-­1β gene is also associated with immune responses to the resistance to F. psychrophilum were not resistant to M. cerebralis. The bacterial pathogen Yersinia ruckeri and involved in resistance to intermediate cross did not appear to be resistant to either pathogen. Aeromonas salmonicida (Baerwald, 2013; Hong et al., 2003; Raida Despite the resistance characteristics of any given trout strain, co- et al., 2011), indicating that achieving an innate response to both infection led to an increase in average mortality for all strains com- pathogens through selective breeding may be possible. In our study, pared to single-­pathogen exposure. we injected F. psychrophilum to ensure that all individuals received It appears that some rainbow trout crosses have greater promise the same dose of bacteria and because immersion exposure to F. for creating dual resistance than others. The results of the second psychrophilum results in considerable variation in exposure depend- experiment indicate that F. psychrophilum-­resistance can be main- ing on experimental circumstances (Avila, 2021; Garcia et al., 2000; tained in first-­generation crosses, with the HL × PRR exhibiting the Langevin et al., 2012). Injection bypasses important innate immune lowest mortality from F. psychrophilum exposure. These crosses may systems of fishes, particularly those in the skin and mucus (Makesh provide another management tool for fisheries managers, similar et al., 2015; Nematollahi et al., 2003), and our results may be influ- to the benefits of using M. cerebralis-­resistant rainbow trout. Use enced by our experimental protocol. Although we choose the injec- of the HL × PRR may reduce or eliminate the need for antibiotics, tion protocol, many of the genes identified in fish immune systems as the probability of mortality from F. psychrophilum exposure was also have a role in acquired fish immune responses (Baerwald, 2013), less than 20%. Additionally, because the Harrison Lake rainbow and it was expected that the immune system would still be activated trout originates from a wild rainbow trout population (Wagner in response to exposure to F. psychrophilum despite the exposure et al., 2006), the HL × PRR may also show better survival and re- method. Clearly, future development of dual resistance will require production after being stocked compared to the PRR, because PRR studies on the immune responses to each pathogen and the activa- are domesticated (Silverstein et al., 2009) and may not do well in tion of these immune responses regarding the order in which expo- the wild. We did not assess how the HL × PRR performed in the sure to each pathogen occurs. presence of M. cerebralis in the second experiment because addi- Although it appears that dual resistance may be possible with tional evaluation was not possible due to time constraints involved some strains, the lack of response in others indicates that dual with the development of the parasite. However, the HL × PRR may resistance may be difficult to develop. The GHP showed no resis- be a good candidate for the development of dual resistance to F. tance to either pathogen as single exposures to F. psychrophilum psychrophilum and M. cerebralis. Prior research has shown that the and dual exposures to F. psychrophilum and M. cerebralis resulted pure HL produce fewer myxospores than we observed in the GHP, in both high mortalities (>75%) and high myxospore counts. High GR × HL, and PRR (Schisler et al., 2011); additional research into M. mortality and high myxospore counts indicate that the GHP is �10 | AVILA et al. not a good candidate for developing dual resistance, particu- the same as those seen in second-­generation backcrosses of F1-­ larly because it seems to have lost resistance to both pathogens. generation GR × CRR with the CRR (Fetherman et al., 2012). A loss of Currently, it is unknown which genes provide resistance to F. resistance could also be the result of the absence of M. cerebralis in psychrophilum (G. Weins, personal communication, February 16, the hatchery system. In a single hatchery generation, the expression 2021). Development of the F. psychrophilum-­resistant rainbow of hundreds of genes in rainbow trout can be altered, resulting in se- trout used selective breeding at the USDA-­A RS NCCWA (Hadidi lection of traits that are beneficial in the hatchery but not in the wild et al., 2008; Leeds et al., 2010; Silverstein et al., 2009; Wiens (Christie et al., 2012, 2016). The absence of the parasite could there- et al., 2013). However, genetic parentage analyses were not done, fore reduce selection for resistance to M. cerebralis given that those and the mechanism of genetic resistance may depend on the spe- genes are not needed for survival in the hatchery environment. The cific parent strains and genes that allow for disease resistance. loss of resistance to M. cerebralis in the GR × HL strain is concerning The GR is highly resistant to M. cerebralis with 9 ± 5 genes esti- for future management and reintroduction efforts. Stocking rainbow mated to confer genetic resistance (Fetherman et al., 2012). These trout that are susceptible to the parasite could result in less success- genes are additive in their effect (Fetherman et al., 2012) and ful survival and recruitment (Avila et al., 2018). Additionally, these therefore if all genes are not passed onto future generations this fish could produce high numbers of myxospores, which may lead to may result in lower resistance to M. cerebralis in outcrosses with increased M. cerebralis in wild systems. F. psychrophilum-­resistant fish. A possible reason that the GHP The PRR strain showed no resistance to M. cerebralis and had 3.45 showed little to no resistance to F. psychrophilum is that the GR times more myxospores than the highly susceptible CRR (Fetherman genes that confer resistance to M. cerebralis may negatively inter- et al., 2011). The GHP had similar numbers of myxospores as the act with the genes that infer F. psychrophilum resistance (Fraslin CRR, also indicating no genetic resistance to M. cerebralis. High et al., 2020; Lhorente et al., 2014). numbers of myxospores and high mortality indicated that neither We included the ARS-­Fp-­R strain to determine if it had higher F. the PRR nor GHP strains are good candidates for stocking into M. psychrophilum resistance than the PRR, because it had undergone cerebralis-­positive waters. Stocking these strains could result in in- more generations of selection (three for the PRR versus five for the creased M. cerebralis and loss of fish due to M. cerebralis infection. ARS-­Fp-­R) and was predicted to show similar or lower mortality due Co-­infection with F. psychrophilum and M. cerebralis increased to the generational differences in F. psychrophilum-­resistance selec- CPM for every rainbow trout strain. Similar increases in mortality tion (G. Weins, personal communication, February 16, 2021). The have been seen with parasite and bacterial co-­infections compared lower mortality in the PRR indicates that it has higher resistance to to single-­pathogen exposure of rainbow trout in previous experi- F. psychrophilum and additional selection in the ARS-­Fp-­R rainbow ments (Bandilla et al., 2006; Busch et al., 2003; Schisler et al., 2000). trout strain did not confer greater resistance. One explanation is that Ma et al. (2019) also showed higher mortality in rainbow trout with there were other environmental variables not accounted for, for ex- co-­infections of F. psychrophilum and infectious hematopoietic ne- ample, transportation of eyed eggs from West Virginia to Colorado crosis virus (IHNV), compared to those infected with a single patho- may have induced additional stress due to temperature and pres- gen. Currently, it is not known what factor(s) increase mortality due sure changes, resulting in increased mortality. Both the ARS-­Fp-­R to co-­infection or the specific interactions between M. cerebralis and and the S-­Line, which were spawned in West Virginia and then sent F. psychrophilum. Co-­infections are common within the hatchery and to Colorado, showed mortality beginning around day two of the wild environments due to exposure to heterogeneous infectious experiment which is slightly earlier than the traditional time frame pathogens (Kotob et al., 2016; Ma et al., 2019), and reduced post-­ seen in all other rainbow trout strains. Another explanation for the stocking survival may result from co-­infection in hatchery or wild differences in mortality between the strains is the continuous ex- environments. Reducing disease exposure in hatcheries by changing posure to F. psychrophilum in the CPW hatchery system that may or improving husbandry protocols may not only reduce disease out- have allowed the PRR to develop increased resistance compared to breaks but increase long-­term survival within the hatchery and/or the ARS-­Fp-­R . Similar continuous exposure is believed to have pro- post-­stocking survival. duced the M. cerebralis genetic resistance in the GR strain (Hedrick Our research demonstrates that the development of dual resis- et al., 2003). Based on these results, there is no need to replace the tance to both F. psychrophilum and M. cerebralis is attainable but current PRR brood stock with another F. psychrophilum-­resistant is dependent on the specific rainbow trout strains that are used, brood stock that experienced more generations of selection in the and presumably the genetic compatibility of their individual resis- CPW hatchery system. tance. The GHP cross was not resistant to either pathogen; how- A concerning and unexpected observation was the relatively high ever, increased resistance to F. psychrophilum in the HL × PRR and average myxospore counts for the M. cerebralis-­resistant GR × HL. HL × ARS-­ Fp-­ R suggests that dual resistance may be possible. The high number of myxospores found in the GR × HL indicates a Further research will be needed to evaluate M. cerebralis resistance loss of resistance and could be attributed to backcrossing or lack of of the HL × PRR or HL × ARS-­Fp-­R crosses. Dual resistance will exposure to the parasite. Outcrossing and/or backcrossing may have benefit both aquaculture production and management of wild fish- occurred in the hatchery and resulted in decreased genetic resis- eries and has implications for the management and protection of tance to M. cerebralis. 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Dual resistance to Flavobacterium psychrophilum and Myxobolus cerebralis in rainbow trout (Oncorhynchus mykiss, Walbaum). Journal of Fish Diseases, 00, 1–­13. https://doi.org/10.1111/jfd.13605 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Journal Articles Description An account of the resource CPW peer-reviewed journal publications Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Dual resistance to Flavobacterium psychrophilum and Myxobolus cerebralis in rainbow trout (Oncorhynchus mykiss, Walbaum) Description An account of the resource Aquatic pathogens are a major concern for fish hatchery production, fisheries management, and conservation, and disease control needs to be addressed. Two important salmonid pathogens are Myxobolus cerebralis and Flavobacterium psychrophilum that cause whirling disease and bacterial coldwater disease (BCWD), respectively. Innate disease resistance is a potential option for reducing disease-related mortality in hatchery-reared rainbow trout (Oncorhynchus mykiss, Walbaum). Two experiments were conducted to assess pathogen resistance of first-generation (F1) rainbow trout created by crossing M. cerebralis- and F. psychrophilum-resistant strains. In the first experiment, we exposed two rainbow trout strains and one F1 cross to six treatments: control (no exposure), mock injection, F. psychrophilum only, M. cerebralis only, F. psychrophilum then M. cerebralis, and M. cerebralis then F. psychrophilum. Results indicated that the F1 cross was not resistant to either pathogen. In the second experiment, we exposed five rainbow trout strains and four rainbow trout crosses to F. psychrophilum. The second experiment indicated that at least one rainbow trout cross was F. psychrophilum-resistant. Achieving dual resistance may be possible using selective breeding but only some multigenerational strains are suitable candidates for further evaluation. Bibliographic Citation A bibliographic reference for the resource. Recommended practice is to include sufficient bibliographic detail to identify the resource as unambiguously as possible. <a href="https://doi.org/10.1111/jfd.13605" target="_blank" rel="noreferrer noopener">https://doi.org/10.1111/jfd.13605</a> Creator An entity primarily responsible for making the resource Avila, Brian W. Winkelman, Dana L. Fetherman, Eric R. Subject The topic of the resource Bacterial coldwater disease <em>Flavobacterium psychrophilum</em> <em>Myxobolus cerebralis</em> Rainbow trout Whirling disease Extent The size or duration of the resource. 13 pages Date Created Date of creation of the resource. 2022-03-08 Rights Information about rights held in and over the resource <a href="http://rightsstatements.org/vocab/InC-NC/1.0/" target="_blank" rel="noreferrer noopener">In Copyright - Non-Commercial Use Permitted</a> <a href="https://creativecommons.org/licenses/by/4.0/" target="_blank" rel="noreferrer noopener">Attribution 4.0 International (CC BY 4.0)</a> Format The file format, physical medium, or dimensions of the resource application/pdf Language A language of the resource English Is Part Of A related resource in which the described resource is physically or logically included. Journal of Fish Diseases Type The nature or genre of the resource Article