doi:10.1111/jfd.12300
Journal of Fish Diseases 2015, 38, 853–857
Short Communication Detection of Ichthyophonus by chromogenic in situ hybridization C M Conway1, M K Purcell1, D G Elliott1 and P K Hershberger2 1 US Geological Survey—Western Fisheries Research Center, Seattle, WA, USA 2 US Geological Survey—Marrowstone Marine Field Station, Nordland, WA, USA
Keywords: Ichthyophonus, mesomycetozoea, ribosomal small subunit DNA. Ichthyophonus hoferi (Plehn & Mulsow 1911) is a protistan parasite in the class Mesomycetozoea that infects a large range of marine and freshwater fish (Mendoza, Taylor & Ajello 2002; McVicar 2011). The broad host and geographic range, which includes both fresh and marine waters of the Northern and Southern Hemispheres, combined with a lack of distinguishing morphological characteristics, have prompted speculation that Ichthyophonus-like organisms in multiple species of fish, as well as reptiles, amphibians, birds and invertebrates, may have been incorrectly classified under a single type species I. hoferi (McVicar 2011). At present, only two species, I. hoferi and I. irregularis, are currently recognized within the genus (Rand et al. 2000; Mendoza et al. 2002). Investigations of ribosomal DNA sequence variation have begun to clarify relationships among Ichthyophonus types (Criscione et al. 2002; Rasmussen et al. 2010). Here, we will use the term Ichthyophonus to broadly represent all members of the genus regardless of species/subspecies. Ichthyophonus disease, or ichthyophoniasis, can result in negative impacts to fisheries by causing recurring epizootics, and declines in affected populations (reviewed in Burge et al. 2014) and by creating skeletal muscle lesions in affected hosts, thereby reducing product quality (reviewed in
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
Correspondence: C M Conway, US Geological Survey- Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115, USA (e-mail: [email protected])
853
McVicar 2011). Because the parasite may reside in both skeletal and cardiac muscle, infection can have significant sublethal effects on growth, condition, reproductive capacity, energy and swimming stamina (Kocan et al. 2006; Kramer-Schadt, Holst & Skagen 2010; Vollenweider et al. 2011). Detection of Ichthyophonus infections typically involves observation of visible signs, including white nodules in the heart, liver, spleen or kidney, combined with culture of explant tissue or microscopic visualization in tissue squash preparations (Kocan, Dolan & Hershberger 2011; Hershberger 2012). Although typically less sensitive than other techniques, histopathology is also widely used and is effective for evaluating Ichthyophonus infections because disease severity and host response can be assessed simultaneously (Kocan et al. 2011). Positive periodic acid-Schiff (PAS) staining of spherical multinucleate organisms 10–250 lm in diameter can be presumptive for Ichthyophonus, but should not be considered confirmatory because a number of PAS-positive organisms occur in this size range (Hershberger 2012). The lack of a definitive Ichthyophonus confirmatory test for histopathological evaluation may lead to misdiagnosis, particularly when the organism cannot be cultured due to lack of material, contamination or inability of the organism to propagate. Infections with fungal organisms may be misidentified as Ichthyophonus due to similar morphological characteristics and histopathological presentation (Sprotson 1944; Maureen Purcell and Paul Hershberger, unpublished results). Conventional and quantitative polymerase chain reaction (PCR)-based confirmation can be
Journal of Fish Diseases 2015, 38, 853–857
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
effective for confirming moderate to heavy infections (Whipps et al. 2006; White et al. 2013), but false-negative results can occur as the parasite is focally distributed and may not be detected if only a small tissue amount is analysed. Additionally, several researchers have reported Ichthyophonus-like organisms in amphibians (McVicar 2011), but it is unclear whether these organisms represent the same species found in fish because they are not culturable and cannot be detected using Ichthyophonus-specific PCR tests (Hershberger & Purcell, unpublished results). In situ hybridization is a method that complements histological diagnosis by providing highly specific molecular confirmation of the observed organism in preserved tissue. Here, we report the development of a chromogenic in situ hybridization (CISH) procedure that specifically detected Ichthyophonus ribosomal DNA in histological sections. Laboratory-reared, specific-pathogen-free Pacific herring, Clupea pallasii Valenciennes, (Hershberger et al. 2007) were infected with Ichthyophonus by intraperitoneal injection. Infected fish and noninfected controls were killed by an overdose of buffered tricaine methanesulfonate (MS-222, Argent Chemical Laboratories) at various days post-injection, and the heart and liver removed. A portion of the tissue was fixed in 10% neutralbuffered formalin (NBF) for 24 or 48 h, transferred to 70% ethanol and then processed and embedded in paraffin wax by conventional histological methods. Formalin-fixed, paraffin-embedded tissues collected from naturally infected Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and red-spotted newt, Notophthalmus viridescens (Rafinesque), and experimentally infected rainbow trout, O. mykiss (Walbaum), and Pacific staghorn sculpin, Leptocottus armatus Girard, were also analysed in this study; those tissues were fixed using various methods. An Ichthyophonus-specific oligonucleotide probe was designed to target conserved portions of the 18S small subunit (SSU) ribosomal gene of known Ichthyophonus species, including I. hoferi (GenBank accession numbers FJ869836, GQ370781, AF467785, U43712, GQ370802, AF467786) and I. irregularis (AF232303); the probe contained nucleotide mismatches to prevent hybridization with closely related mesomycetozoan species (Rhinosporidium seeberi, AF118851; Dermocystidium sp., AF533950; Paramoebidium sp., AY336708; Amoebidium parasiticum, Y19155). The digoxigenin (DIG)-labelled probe (50 -/ 5DigN/GCC TTC GAG AAG AAG AAA CTG -30 ) 854
C M Conway et al. Ichthyophonus CISH
was commercially synthesized (Integrated DNA Technologies, Inc.). The Ichthyophonus CISH hybridization protocol was modified from previously reported methods (McCarthy, Urquhart & Bricknell 2008; Marcino 2013). All steps were performed at room temperature (22–25 °C) unless otherwise specified (Table 1). Briefly, 5-lm tissue sections were adhered to positively charged glass slides (Colorfrostâ Plus; Fisher Scientific), dried and dewaxed. Tissues were then rehydrated through a graded ethanol series followed by deionized water. Sections were equilibrated in phosphate-buffered saline and then permeabilized with proteinase K in a humid chamber at 37 °C for 40 min. Proteolysis was stopped by immersing sections in 0.2% glycine, followed by immersion in glacial acetic acid and washes in Tris-buffered saline and 59 SET buffer. A hybridization chamber (HybriWell-FL; Sigma-Aldrich) was applied to each slide and filled with pre-warmed (42 °C) hybridization solution before incubating in a humid chamber at 42 °C for 90 min. The hybridization solution was removed and replaced with the Ichthyophonus probe diluted to 6 ng lL1 in 42 °C hybridization solution and incubated in a humid chamber at 42 °C overnight (18–20 h). Hybridization chambers were removed, and slides immersed in 42 °C 0.29 SET and then in Buffer 1. Sections were then immersed in blocking solution for 1 h. New hybridization chambers were applied to slides, filled with diluted anti-DIG-alkaline phosphatase conjugate solution and then incubated in a humid chamber for 3 h. Sections were then washed in Buffer 1, followed by Buffer 2. The colour development solution was prepared, and sections were immediately immersed and incubated for 2 h in the dark, followed by washing in TE buffer and then deionized water. Bleaching of endogenous melanin was performed after the CISH procedure; sections were immersed in 0.25% potassium permanganate for 15 min, rinsed in deionized water, immersed in 5% oxalic acid for 5 min and rinsed again in deionized water. Slides were mounted with aqueous mounting medium and glass coverslips. Serial sections were stained by PAS following standard methods (Carson 1997). Hybridization of the DIG-labelled probe to Ichthyophonus nucleic acid was indicated by dark purple precipitates in schizonts and other developmental stages (Figs 1a and c) and
Journal of Fish Diseases 2015, 38, 853–857
C M Conway et al. Ichthyophonus CISH
Table 1 Chromogenic in situ hybridization protocol for the detection of Ichthyophonus in formalin-fixed, paraffin-embedded tissuesa In situ Hybridization Step Dewaxing and Rehydration
Permeabilization
Prehybridization
Hybridization
Post-hybridization washes Hybridization detection
Melanin bleach
Mount
Reagent
Time TM
Clear-Rite 3 (ThermoFisher Scientific) 100% and 95% ethanol 80%, 70%, 50% and 30% ethanol Deionized water Phosphate-buffered saline (PBS): 0.014 M, pH 7.4 Proteinase K (Sigma-Aldrich P2308): 25 lg mLa in 37 °C PBS Incubate in a humid chamber 0.2% glycine (w/v in PBS) Glacial acetic acid Tris-buffered saline (TBS): 0.05 M Tris, 0.15 M NaCl; pH 7.6 59 SET buffer: 0.75 M NaCl, 0.0064 M EDTA, 0.10 M Tris; pH 8.0 Hybridization solution (42 °C): 59 SET, 0.020% [w/v] bovine serum albumin, 0.025% [w/v] sodium dodecyl sulphate Apply HybriWell-FLTM hybridization chamber (Sigma-Aldrich), add 200 lL of 42 °C hybridization solution. Incubate in a humid chamber Dilute probe to 6 ng lL1 in 42 °C hybridization solution Replace hybridization solution with 200 lL of diluted probe Incubate in a humid chamber Remove hybridization chamber 42 °C 0.29 SET Buffer 1: 0.10 M Tris, 0.15 M NaCl; pH 7.5 Blocking solution: Buffer 1, 0.3% Triton Xâ-100, 2% normal sheep serum (NSS). Incubate in a plastic 5-slide mailer Anti-DIG-alkaline phosphatase (AP) conjugate solution (Roche Applied Science) diluted 1:500 in Buffer 1, 0.3% Triton Xâ-100 and 1% NSS. Apply a new hybridization chamber; add 200 lL of conjugate solution. Incubate in a humid chamber Remove hybridization chamber Buffer 1 Buffer 2: 0.10 M Tris, 0.10 M NaCl, 0.05 M MgCl2; pH 9.5 Colour development solution: Buffer 2, 0.45% NBT [v/v] 0.35% BCIP [v/v], 1% levamisole (w/v) 24 mg mL1 Prepare immediately before use; incubate in a plastic 5-slide mailer TE buffer: 0.01 M Tris pH 8.0, 0.001 M EDTA Deionized water 0.25% potassium permanganate Deionized water 5% oxalic acid Deionized water Mount coverglass with Faramount aqueous mounting medium (Dako)
2x, 5 min 2x, 3 min 1x, 3 min 3x, 1 min 5 min 40 min at 37°C 2x, 5 min 30 s 5 min 2x, 5 min 90 min at 42°C
18–20 h at 42°C
2x, 5 min 5 min 1h 3h
2x, 5 min 2x, 5 min 2 h in the dark
5 min 2x, 3 min 15 min 30 s 5 min 30 s
a Section tissues at 4–5 lm and mount on positively charged glass slides. All steps are performed at room temperature (22–25 °C) unless otherwise stated. The number of solution changes is noted by ‘x’.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
correlated with the distribution and morphology of cells observed in PAS-stained tissues (Figs 1b and d). Further, the CISH procedures were effective at identifying Ichthyophonus developmental stages in locations where the parasite was in the presence of normal host tissues staining PAS positive (Fig. 1d). No hybridization signals or evidence of Ichthyophonus infection were detected in specific-pathogen-free Pacific herring control tissues by CISH or PAS (photographs not shown). Ichthyophonus nucleic acid was successfully detected in Pacific herring (Fig. 1a), rainbow trout (Fig. 1c), Pacific staghorn sculpin and Chinook salmon (photographs not shown). Genetic typing of Ichthyophonus from the North American West 855
Coast indicates that Ichthyophonus from freshwater rainbow trout is a genetically distinct type compared with the more broadly distributed type infecting marine and anadromous fish species in the region (Rasmussen et al. 2010). We observed no detectable difference in CISH hybridization quality between Ichthyophonus genetic types or between laboratory-challenged and naturally occurring infections (photographs not shown). An Ichthyophonus-like, PAS-positive organism in redspotted newt tissues (Fig. 1f) did not hybridize with the Ichthyophonus probe (Fig. 1e). This result supports the hypothesis that the organism infecting amphibians is taxonomically distinct from fish-associated Ichthyophonus.
Journal of Fish Diseases 2015, 38, 853–857
(a)
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
(b)
(c)
(d)
(e)
(f)
There was no difference in dewaxing efficiency between Clear-RiteTM 3 (ThermoFisher Scientific) and xylene; however, several schizonts detached from slides when the total dewaxing time exceeded 10 min. The addition of conventional denaturation and annealing steps did not improve the sensitivity of Ichthyophonus nucleic acid detection; however, incorporation of these steps did result in increased degradation of fish tissues and parasite schizonts. Several different counter stains were tested during development of the CISH protocol, but these stains did not improve visualization of cellular morphology. However, melanin bleaching was required to confidently distinguish positive CISH staining from tissue melanin. In summary, we developed a CISH assay capable of detecting Ichthyophonus nucleic acid in standard histology sections. The assay has utility for both diagnostic and research applications. The CISH assay can be used to confirm histological diagnosis of Ichthyophonus or be readily applied to archival material. There remain many unanswered questions regarding the Ichthyophonus life cycle and transmission routes. For example, although Ichthyophonus is typically observed as 10- to 250856
C M Conway et al. Ichthyophonus CISH
Figure 1 Comparisons between the newly developed chromogenic in situ hybridization (CISH) procedure and conventional periodic acid-Schiff (PAS) staining for Ichthyophonus detection in histological sections. Ichthyophonus schizonts and other developmental stages were easily identified by both CISH and PAS in an epicardial granuloma in Pacific herring (a and b, respectively). CISH was effective at identifying Ichthyophonus stages in rainbow trout stomach tunica propria (c), where the parasite was in the presence of PAS-positive host tissues (d). Parasitic stages in the skeletal muscle of red-spotted newt, previously identified as Ichthyophonus, failed to hybridize with the Ichthyophonus-specific probe (e) (section counter-stained with Bismarck brown Y for visibility), but stained PAS positive (f). Scale bars = 100 lm.
lm-diameter schizonts in tissues of live fishes, the parasitic life stages can be extremely pleomorphic in vitro and additional life stages have been reported in vivo (Kocan, LaPatra & Hershberger 2013), raising the possibility that small cryptic stages of the parasite have been overlooked when non-specific histological stains are used. If this is the case, the CISH assay may be useful for tracking sequential dissemination of Ichthyophonus throughout fish tissues following laboratory exposure. Additionally, the CISH assay may prove beneficial in ongoing studies intended to identify non-piscine intermediate hosts for the parasite. Acknowledgements The authors acknowledge the assistance of Joe Marcino (Maryland Department of Natural Resources), Ryan Carnegie and Nancy Stokes (Virginia Institute of Marine Sciences), Rod Getchell (Cornell University) and Richard Kocan (University of Washington). Funding was provided by the Exxon Valdez Oil Spill Trustee Council, Project # 10100132-I and the Fisheries Program of the Ecosystem Mission area of the
Journal of Fish Diseases 2015, 38, 853–857
U.S. Geological Survey. All animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Western Fisheries Research Center. The use of trade, firm or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of the Interior or the U.S. Geological Survey of any product or service to the exclusion of others that may be suitable. Publication History Received: 25 April 2014 Revision received: 10 July 2014 Accepted: 21 July 2014 This paper was edited and accepted under the Editorship of Professor Ron Roberts.
References Burge C.A., Eakin C.M., Friedman C.S., Froelich B., Hershberger P.K., Hofmann E.E., Petes L.E., Prager K.C., Weil E., Willis B.L., Ford S.E. & Harvell C.D. (2014) Climate change influences on marine infectious diseases: implications for management and society. Annual Review of Marine Science 6, 249–277. Carson F.L. (1997) In: Histotechnology, A Self-Instructional Text, 2nd edn, pp. 113–114. American Society of Clinical Pathologists, Chicago, IL. Criscione C.D., Watral V., Whipps C.M., Blouin M.S., Jones S.R.M. & Kent M.L. (2002) Ribosomal DNA sequences indicate isolated populations of Ichthyophonus hoferi in geographic sympatry in the north-eastern Pacific Ocean. Journal of Fish Diseases 25, 575–582. Hershberger P.K. (2012) Ichthyophonus Disease (Ichthyophoniasis). Fish Health Section Blue Book: Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens, pp. 1–13. American Fisheries Society, Bethesda, MD. Hershberger P.K., Gregg J., Pacheco C., Winton J., Richard J. & Traxler G. (2007) Larval Pacific herring, Clupea pallasii (Valenciennes), are highly susceptible to viral haemorrhagic septicaemia and survivors are partially protected after their metamorphosis to juveniles. Journal of Fish Diseases 30, 445–458. Kocan R., LaPatra S., Gregg J., Winton J. & Hershberger P. (2006) Ichthyophonus-induced cardiac damage: a mechanism for reduced swimming stamina in salmonids. Journal of Fish Diseases 29, 521–527.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
Kocan R., Dolan H. & Hershberger P. (2011) Diagnostic methodology is critical for accurately determining the prevalence of Ichthyophonus infections in wild fish populations. Journal of Parasitology 97, 344–348.
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Kocan R., LaPatra S. & Hershberger P. (2013) Evidence for an amoeba-like infectious stage of Ichthyophonus sp. and description of a circulating blood stage: a probable mechanism for dispersal within the fish host. Journal of Parasitology 99, 235–240. Kramer-Schadt S., Holst J.C. & Skagen D. (2010) Analysis of variables associated with the Ichthyophonus hoferi epizootics in Norwegian spring spawning herring, 1992-2008. Canadian Journal of Fisheries and Aquatic Sciences 67, 1862– 1873. Marcino J. (2013) A comparison of two methods for colorimetric in situ hybridization using paraffinembedded tissue sections and digoxigenin-labeled hybridization probes. Journal of Aquatic Animal Health 25, 119–124. McCarthy U.M., Urquhart K.L. & Bricknell I.R. (2008) An improved in situ hybridization method for the detection of fish pathogens. Journal of Fish Diseases 31, 669–677. McVicar A.H. (2011) Ichthyophonus. In: Fish Diseases and Disorders, Vol. 3: Viral, Bacterial, and Fungal Infections 2nd edn. (ed. by P.T.K. Woo & D.W. Bruno) pp. 721–747. Cambridge, MA, CABI. Wallingford, UK. Mendoza L., Taylor J.W. & Ajello L. (2002) The class mesomycetozoea: a heterogeneous group of microorganisms at the animal-fungal boundary. Annual Review of Microbiology 56, 315–344. Plehn M. & Mulsow K. (1911) Der erreger der ‘Taummelkrankheit’ der salmoiden. Zentralblatt f€ ur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene 59, 63–68. Rand T.G., White K., Cannone J.J., Gutell R.R., Murphy C.A. & Ragan M.A. (2000) Ichthyophonus irregularis sp. nov. from the yellowtail flounder Limanda ferruginea from the Nova Scotia shelf. Diseases of Aquatic Organisms 41, 31–36. Rasmussen C., Purcell M.K., Gregg J.L., LaPatra S.E., Winton J.R. & Hershberger P.K. (2010) Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout. Diseases of Aquatic Organisms 89, 179–183. Sprotson N.G. (1944) Ichthyosporidium hoferi (Plehn and Mulsow, 1911), an internal fungoid parasite of the mackerel. Journal of the Marine Biological Association of the United Kingdom 26, 72–98. Vollenweider J., Gregg J., Heintz R. & Hershberger P. (2011) Energetic cost of Ichthyophonus infection in juvenile Pacific herring (Clupea pallasii). Journal of Parasitology Research 926812, 10. Whipps C.M., Burton T., Watral V.G., St-Hilaire S. & Kent M.L. (2006) Assessing the accuracy of a polymerase chain reaction test for Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha. Diseases of Aquatic Organisms 68, 141–147. White V.C., Morado J.F., Crosson L.M., Vadopalas B. & Friedman C.S. (2013) Development and validation of a quantitative PCR assay for Ichthyophonus spp. Diseases of Aquatic Organisms 104, 69–81.
Journal of Fish Diseases 2015, 38, 853–857
Short Communication Detection of Ichthyophonus by chromogenic in situ hybridization C M Conway1, M K Purcell1, D G Elliott1 and P K Hershberger2 1 US Geological Survey—Western Fisheries Research Center, Seattle, WA, USA 2 US Geological Survey—Marrowstone Marine Field Station, Nordland, WA, USA
Keywords: Ichthyophonus, mesomycetozoea, ribosomal small subunit DNA. Ichthyophonus hoferi (Plehn & Mulsow 1911) is a protistan parasite in the class Mesomycetozoea that infects a large range of marine and freshwater fish (Mendoza, Taylor & Ajello 2002; McVicar 2011). The broad host and geographic range, which includes both fresh and marine waters of the Northern and Southern Hemispheres, combined with a lack of distinguishing morphological characteristics, have prompted speculation that Ichthyophonus-like organisms in multiple species of fish, as well as reptiles, amphibians, birds and invertebrates, may have been incorrectly classified under a single type species I. hoferi (McVicar 2011). At present, only two species, I. hoferi and I. irregularis, are currently recognized within the genus (Rand et al. 2000; Mendoza et al. 2002). Investigations of ribosomal DNA sequence variation have begun to clarify relationships among Ichthyophonus types (Criscione et al. 2002; Rasmussen et al. 2010). Here, we will use the term Ichthyophonus to broadly represent all members of the genus regardless of species/subspecies. Ichthyophonus disease, or ichthyophoniasis, can result in negative impacts to fisheries by causing recurring epizootics, and declines in affected populations (reviewed in Burge et al. 2014) and by creating skeletal muscle lesions in affected hosts, thereby reducing product quality (reviewed in
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
Correspondence: C M Conway, US Geological Survey- Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115, USA (e-mail: [email protected])
853
McVicar 2011). Because the parasite may reside in both skeletal and cardiac muscle, infection can have significant sublethal effects on growth, condition, reproductive capacity, energy and swimming stamina (Kocan et al. 2006; Kramer-Schadt, Holst & Skagen 2010; Vollenweider et al. 2011). Detection of Ichthyophonus infections typically involves observation of visible signs, including white nodules in the heart, liver, spleen or kidney, combined with culture of explant tissue or microscopic visualization in tissue squash preparations (Kocan, Dolan & Hershberger 2011; Hershberger 2012). Although typically less sensitive than other techniques, histopathology is also widely used and is effective for evaluating Ichthyophonus infections because disease severity and host response can be assessed simultaneously (Kocan et al. 2011). Positive periodic acid-Schiff (PAS) staining of spherical multinucleate organisms 10–250 lm in diameter can be presumptive for Ichthyophonus, but should not be considered confirmatory because a number of PAS-positive organisms occur in this size range (Hershberger 2012). The lack of a definitive Ichthyophonus confirmatory test for histopathological evaluation may lead to misdiagnosis, particularly when the organism cannot be cultured due to lack of material, contamination or inability of the organism to propagate. Infections with fungal organisms may be misidentified as Ichthyophonus due to similar morphological characteristics and histopathological presentation (Sprotson 1944; Maureen Purcell and Paul Hershberger, unpublished results). Conventional and quantitative polymerase chain reaction (PCR)-based confirmation can be
Journal of Fish Diseases 2015, 38, 853–857
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
effective for confirming moderate to heavy infections (Whipps et al. 2006; White et al. 2013), but false-negative results can occur as the parasite is focally distributed and may not be detected if only a small tissue amount is analysed. Additionally, several researchers have reported Ichthyophonus-like organisms in amphibians (McVicar 2011), but it is unclear whether these organisms represent the same species found in fish because they are not culturable and cannot be detected using Ichthyophonus-specific PCR tests (Hershberger & Purcell, unpublished results). In situ hybridization is a method that complements histological diagnosis by providing highly specific molecular confirmation of the observed organism in preserved tissue. Here, we report the development of a chromogenic in situ hybridization (CISH) procedure that specifically detected Ichthyophonus ribosomal DNA in histological sections. Laboratory-reared, specific-pathogen-free Pacific herring, Clupea pallasii Valenciennes, (Hershberger et al. 2007) were infected with Ichthyophonus by intraperitoneal injection. Infected fish and noninfected controls were killed by an overdose of buffered tricaine methanesulfonate (MS-222, Argent Chemical Laboratories) at various days post-injection, and the heart and liver removed. A portion of the tissue was fixed in 10% neutralbuffered formalin (NBF) for 24 or 48 h, transferred to 70% ethanol and then processed and embedded in paraffin wax by conventional histological methods. Formalin-fixed, paraffin-embedded tissues collected from naturally infected Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and red-spotted newt, Notophthalmus viridescens (Rafinesque), and experimentally infected rainbow trout, O. mykiss (Walbaum), and Pacific staghorn sculpin, Leptocottus armatus Girard, were also analysed in this study; those tissues were fixed using various methods. An Ichthyophonus-specific oligonucleotide probe was designed to target conserved portions of the 18S small subunit (SSU) ribosomal gene of known Ichthyophonus species, including I. hoferi (GenBank accession numbers FJ869836, GQ370781, AF467785, U43712, GQ370802, AF467786) and I. irregularis (AF232303); the probe contained nucleotide mismatches to prevent hybridization with closely related mesomycetozoan species (Rhinosporidium seeberi, AF118851; Dermocystidium sp., AF533950; Paramoebidium sp., AY336708; Amoebidium parasiticum, Y19155). The digoxigenin (DIG)-labelled probe (50 -/ 5DigN/GCC TTC GAG AAG AAG AAA CTG -30 ) 854
C M Conway et al. Ichthyophonus CISH
was commercially synthesized (Integrated DNA Technologies, Inc.). The Ichthyophonus CISH hybridization protocol was modified from previously reported methods (McCarthy, Urquhart & Bricknell 2008; Marcino 2013). All steps were performed at room temperature (22–25 °C) unless otherwise specified (Table 1). Briefly, 5-lm tissue sections were adhered to positively charged glass slides (Colorfrostâ Plus; Fisher Scientific), dried and dewaxed. Tissues were then rehydrated through a graded ethanol series followed by deionized water. Sections were equilibrated in phosphate-buffered saline and then permeabilized with proteinase K in a humid chamber at 37 °C for 40 min. Proteolysis was stopped by immersing sections in 0.2% glycine, followed by immersion in glacial acetic acid and washes in Tris-buffered saline and 59 SET buffer. A hybridization chamber (HybriWell-FL; Sigma-Aldrich) was applied to each slide and filled with pre-warmed (42 °C) hybridization solution before incubating in a humid chamber at 42 °C for 90 min. The hybridization solution was removed and replaced with the Ichthyophonus probe diluted to 6 ng lL1 in 42 °C hybridization solution and incubated in a humid chamber at 42 °C overnight (18–20 h). Hybridization chambers were removed, and slides immersed in 42 °C 0.29 SET and then in Buffer 1. Sections were then immersed in blocking solution for 1 h. New hybridization chambers were applied to slides, filled with diluted anti-DIG-alkaline phosphatase conjugate solution and then incubated in a humid chamber for 3 h. Sections were then washed in Buffer 1, followed by Buffer 2. The colour development solution was prepared, and sections were immediately immersed and incubated for 2 h in the dark, followed by washing in TE buffer and then deionized water. Bleaching of endogenous melanin was performed after the CISH procedure; sections were immersed in 0.25% potassium permanganate for 15 min, rinsed in deionized water, immersed in 5% oxalic acid for 5 min and rinsed again in deionized water. Slides were mounted with aqueous mounting medium and glass coverslips. Serial sections were stained by PAS following standard methods (Carson 1997). Hybridization of the DIG-labelled probe to Ichthyophonus nucleic acid was indicated by dark purple precipitates in schizonts and other developmental stages (Figs 1a and c) and
Journal of Fish Diseases 2015, 38, 853–857
C M Conway et al. Ichthyophonus CISH
Table 1 Chromogenic in situ hybridization protocol for the detection of Ichthyophonus in formalin-fixed, paraffin-embedded tissuesa In situ Hybridization Step Dewaxing and Rehydration
Permeabilization
Prehybridization
Hybridization
Post-hybridization washes Hybridization detection
Melanin bleach
Mount
Reagent
Time TM
Clear-Rite 3 (ThermoFisher Scientific) 100% and 95% ethanol 80%, 70%, 50% and 30% ethanol Deionized water Phosphate-buffered saline (PBS): 0.014 M, pH 7.4 Proteinase K (Sigma-Aldrich P2308): 25 lg mLa in 37 °C PBS Incubate in a humid chamber 0.2% glycine (w/v in PBS) Glacial acetic acid Tris-buffered saline (TBS): 0.05 M Tris, 0.15 M NaCl; pH 7.6 59 SET buffer: 0.75 M NaCl, 0.0064 M EDTA, 0.10 M Tris; pH 8.0 Hybridization solution (42 °C): 59 SET, 0.020% [w/v] bovine serum albumin, 0.025% [w/v] sodium dodecyl sulphate Apply HybriWell-FLTM hybridization chamber (Sigma-Aldrich), add 200 lL of 42 °C hybridization solution. Incubate in a humid chamber Dilute probe to 6 ng lL1 in 42 °C hybridization solution Replace hybridization solution with 200 lL of diluted probe Incubate in a humid chamber Remove hybridization chamber 42 °C 0.29 SET Buffer 1: 0.10 M Tris, 0.15 M NaCl; pH 7.5 Blocking solution: Buffer 1, 0.3% Triton Xâ-100, 2% normal sheep serum (NSS). Incubate in a plastic 5-slide mailer Anti-DIG-alkaline phosphatase (AP) conjugate solution (Roche Applied Science) diluted 1:500 in Buffer 1, 0.3% Triton Xâ-100 and 1% NSS. Apply a new hybridization chamber; add 200 lL of conjugate solution. Incubate in a humid chamber Remove hybridization chamber Buffer 1 Buffer 2: 0.10 M Tris, 0.10 M NaCl, 0.05 M MgCl2; pH 9.5 Colour development solution: Buffer 2, 0.45% NBT [v/v] 0.35% BCIP [v/v], 1% levamisole (w/v) 24 mg mL1 Prepare immediately before use; incubate in a plastic 5-slide mailer TE buffer: 0.01 M Tris pH 8.0, 0.001 M EDTA Deionized water 0.25% potassium permanganate Deionized water 5% oxalic acid Deionized water Mount coverglass with Faramount aqueous mounting medium (Dako)
2x, 5 min 2x, 3 min 1x, 3 min 3x, 1 min 5 min 40 min at 37°C 2x, 5 min 30 s 5 min 2x, 5 min 90 min at 42°C
18–20 h at 42°C
2x, 5 min 5 min 1h 3h
2x, 5 min 2x, 5 min 2 h in the dark
5 min 2x, 3 min 15 min 30 s 5 min 30 s
a Section tissues at 4–5 lm and mount on positively charged glass slides. All steps are performed at room temperature (22–25 °C) unless otherwise stated. The number of solution changes is noted by ‘x’.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
correlated with the distribution and morphology of cells observed in PAS-stained tissues (Figs 1b and d). Further, the CISH procedures were effective at identifying Ichthyophonus developmental stages in locations where the parasite was in the presence of normal host tissues staining PAS positive (Fig. 1d). No hybridization signals or evidence of Ichthyophonus infection were detected in specific-pathogen-free Pacific herring control tissues by CISH or PAS (photographs not shown). Ichthyophonus nucleic acid was successfully detected in Pacific herring (Fig. 1a), rainbow trout (Fig. 1c), Pacific staghorn sculpin and Chinook salmon (photographs not shown). Genetic typing of Ichthyophonus from the North American West 855
Coast indicates that Ichthyophonus from freshwater rainbow trout is a genetically distinct type compared with the more broadly distributed type infecting marine and anadromous fish species in the region (Rasmussen et al. 2010). We observed no detectable difference in CISH hybridization quality between Ichthyophonus genetic types or between laboratory-challenged and naturally occurring infections (photographs not shown). An Ichthyophonus-like, PAS-positive organism in redspotted newt tissues (Fig. 1f) did not hybridize with the Ichthyophonus probe (Fig. 1e). This result supports the hypothesis that the organism infecting amphibians is taxonomically distinct from fish-associated Ichthyophonus.
Journal of Fish Diseases 2015, 38, 853–857
(a)
Published 2014. This article is a U.S. Government work and is in the public domain in the USA
(b)
(c)
(d)
(e)
(f)
There was no difference in dewaxing efficiency between Clear-RiteTM 3 (ThermoFisher Scientific) and xylene; however, several schizonts detached from slides when the total dewaxing time exceeded 10 min. The addition of conventional denaturation and annealing steps did not improve the sensitivity of Ichthyophonus nucleic acid detection; however, incorporation of these steps did result in increased degradation of fish tissues and parasite schizonts. Several different counter stains were tested during development of the CISH protocol, but these stains did not improve visualization of cellular morphology. However, melanin bleaching was required to confidently distinguish positive CISH staining from tissue melanin. In summary, we developed a CISH assay capable of detecting Ichthyophonus nucleic acid in standard histology sections. The assay has utility for both diagnostic and research applications. The CISH assay can be used to confirm histological diagnosis of Ichthyophonus or be readily applied to archival material. There remain many unanswered questions regarding the Ichthyophonus life cycle and transmission routes. For example, although Ichthyophonus is typically observed as 10- to 250856
C M Conway et al. Ichthyophonus CISH
Figure 1 Comparisons between the newly developed chromogenic in situ hybridization (CISH) procedure and conventional periodic acid-Schiff (PAS) staining for Ichthyophonus detection in histological sections. Ichthyophonus schizonts and other developmental stages were easily identified by both CISH and PAS in an epicardial granuloma in Pacific herring (a and b, respectively). CISH was effective at identifying Ichthyophonus stages in rainbow trout stomach tunica propria (c), where the parasite was in the presence of PAS-positive host tissues (d). Parasitic stages in the skeletal muscle of red-spotted newt, previously identified as Ichthyophonus, failed to hybridize with the Ichthyophonus-specific probe (e) (section counter-stained with Bismarck brown Y for visibility), but stained PAS positive (f). Scale bars = 100 lm.
lm-diameter schizonts in tissues of live fishes, the parasitic life stages can be extremely pleomorphic in vitro and additional life stages have been reported in vivo (Kocan, LaPatra & Hershberger 2013), raising the possibility that small cryptic stages of the parasite have been overlooked when non-specific histological stains are used. If this is the case, the CISH assay may be useful for tracking sequential dissemination of Ichthyophonus throughout fish tissues following laboratory exposure. Additionally, the CISH assay may prove beneficial in ongoing studies intended to identify non-piscine intermediate hosts for the parasite. Acknowledgements The authors acknowledge the assistance of Joe Marcino (Maryland Department of Natural Resources), Ryan Carnegie and Nancy Stokes (Virginia Institute of Marine Sciences), Rod Getchell (Cornell University) and Richard Kocan (University of Washington). Funding was provided by the Exxon Valdez Oil Spill Trustee Council, Project # 10100132-I and the Fisheries Program of the Ecosystem Mission area of the
Journal of Fish Diseases 2015, 38, 853–857
U.S. Geological Survey. All animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Western Fisheries Research Center. The use of trade, firm or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of the Interior or the U.S. Geological Survey of any product or service to the exclusion of others that may be suitable. Publication History Received: 25 April 2014 Revision received: 10 July 2014 Accepted: 21 July 2014 This paper was edited and accepted under the Editorship of Professor Ron Roberts.
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