Detection of the myxosporean parasite Parvicapsula pseudobranchicola in Atlantic salmon (Salmo salar L.) using in situ hybridization (ISH)

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• 作者：Turhan Markussen (1)
  Celia Agusti (1) (5)
  Egil Karlsbakk (2)
  Are Nylund (3)
  脴yvind Brevik (4)
  Haakon Hansen (1)
  
  1. Norwegian Veterinary Institute ; P.O. Box 750 Sentrum ; N-0106 ; Oslo ; Norway
  5. Present address ; Institute of Food Safety and Infection Biology ; Norwegian University of Life Sciences ; P.O. Box 8146 ; Dep. ; NO-0033 ; Oslo ; Norway
  2. Institute of Marine Research ; P.O. Box 1870 ; Nordnes ; 5817 ; Bergen ; Norway
  3. Department of Biology ; University of Bergen ; Thorm酶hlensgt 55 ; N-5020 ; Bergen ; Norway
  4. Cermaq Group AS ; Dronning Eufemias gate 16 ; N-0191 ; Oslo ; Norway
  
• 关键词：Parvicapsula pseudobranchicola ; Myxozoa ; Atlantic salmon ; Parasites ; in situ hybridization ; ISH ; Real ; time PCR ; Locked nucleic acid probe ; LNA probe
• 刊名：Parasites & Vectors
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：8
• 期：1
• 全文大小：1,930 KB
• 参考文献：1. Nylund A, Karlsbakk E, S忙ther PA, Koren C, Larsen T, Nielsen BD, et al. / Parvicapsula pseudobranchicola (Myxosporea) in farmed Atlantic salmon / Salmo salar: tissue distribution, diagnosis and phylogeny. Dis Aquat Org. 2005;63:197鈥?04. CrossRef
  2. Karlsbakk E, J酶rgensen A, Nikolaisen V, Aleksandersen S, Ottem KF, Nylund A. Parvicapsulosis in Farmed Atlantic Salmon. In: / Fisken og Havet (Havforskningsrapporten s忙rnummer I). 2010. p. 105鈥?. in Norwegian.
  3. J酶rgensen A, Nylund A, Nikolaisen V, Alexandersen S, Karlsbakk E. Real-time PCR detection of / Parvicapsula pseudobranchicola (Myxozoa: Myxosporea) in wild salmonids in Norway. J Fish Dis. 2011;34:365鈥?1. CrossRef
  4. Karlsbakk E, S忙ter PA, H酶stlund C, Fjells酶y KR, Nylund A. / Parvicapsula pseudobranchicola n.sp. (Myxozoa), a myxosporidian infecting the pseudobranch of cultured Atlantic salmon ( / Salmo salar) in Norway. Bull Eur Assoc Fish Pathol. 2002;22:381鈥?.
  5. Sterud E, Simolin P, Kvellestad A. Infection by / Parvicapsula sp. (Myxozoa) is associated with mortality in sea-caged Atlantic salmon / Salmo salar in northern Norway. Dis Aquat Org. 2003;54:259鈥?3. CrossRef
  6. M枚lich A, Waser W, Heisler N. The teleost pseudobranch: a role for preconditioning of ocular blood supply? Fish Physiol Biochem. 2009;35:273鈥?6. CrossRef
  7. Bartholomew JL, Atkinson SD, Hallett SL. Involvement of / Manayunkia speciosa (Annelida: Polychaeta: Sabellidae) in the life cycle of / Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon. J Parasitol. 2006;92:742鈥?. CrossRef
  8. K酶ie M, Karlsbakk E, Nylund A. A new genus / Gadimyxa with three new species (Myxozoa, Parvicapsulidae) parasitic in marine fish (Gadidae) and the two-host life cycle of / Gadimyxa atlantica n. sp. J Parasitol. 2007;93:1459鈥?7. CrossRef
  9. K酶ie M, Karlsbakk E, Einen AC, Nylund A. A parvicapsulid (Myxozoa) infecting / Sprattus sprattus and / Clupea harengus (Clupeidae) in the Northeast Atlantic uses / Hydroides norvegicus (Serpulidae) as invertebrate host. Folia Parasitol (Praha). 2013;60:149鈥?4. CrossRef
  10. Morris DJ, Adams A, Richards RH. / In situ hybridisation identifies the gill as a portal of entry for PKX (Phylum Myxozoa), the causative agent of proliferative kidney disease in salmonids. Parasitol Res. 2000;86:950鈥?. CrossRef
  11. Holzer AS, Sommerville C, Wootten R. Tracing the route of / Sphaerospora truttae from the entry locus to the target organ of the host, / Salmo salar L., using an optimized and specific / in situ hybridization technique. J Fish Dis. 2003;26:647鈥?5. CrossRef
  12. Bjork SJ, Bartholomew JL. Invasion of / Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts. Int J Parasitol. 2010;40:1087鈥?5. CrossRef
  13. Holzer AS, Bartosova P, Peckova H, Tyml T, Atkinson S, Bartholomew J, et al. 鈥榃ho鈥檚 who鈥?in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish鈥搈olecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto. Parasitology. 2013;140:46鈥?0. CrossRef
  14. Atkinson SD, Foott JS, Bartholomew JL. Erection of / Ceratonova n. gen. (Myxosporea: Ceratomyxidae) to encompass freshwater species / C. gasterostea n. sp. from threespine stickleback ( / Gasterosteus aculeatus) and / C. shasta n. comb. from salmonid fishes. J Parasitol. 2014;100:640鈥?. CrossRef
  15. Holzer AS, Sommerville C, Wootten R. Molecular studies on the seasonal occurrence and development of five myxozoans in farmed / Salmo trutta L. Parasitology. 2006;132:193鈥?05. CrossRef
  16. Kelley GO, Zagmutt-Vergara FJ, Leutenegger CM, Adkison MA, Baxa DV, Hedrick RP. Identification of a serine protease gene expressed by / Myxobolus cerebralis during development in rainbow trout / Oncorhynchus mykiss. Dis Aquat Org. 2004;59:235鈥?8. CrossRef
  17. Nylund S, Andersen L, Saevareid I, Plarre H, Watanabe K, Arnesen CE, et al. Diseases of farmed Atlantic salmon / Salmo salar associated with infections by the microsporidian / Paranucleospora theridion. Dis Aquat Org. 2011;94:41鈥?7. CrossRef
  18. Petersen M, Wengel J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 2003;21:74鈥?1. CrossRef
  19. Thomsen R, Nielsen PS, Jensen TH. Dramatically improved RNA / in situ hybridization signals using LNA-modified probes. RNA. 2005;11:1745鈥?. CrossRef
  20. Kubota K, Ohashi A, Imachi H, Harada H. Improved / in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes. Appl Environ Microbiol. 2006;72:5311鈥?. CrossRef
  21. Holzer AS, Stewart S, Tildesley A, Wootten R, Sommerville C. Infection dynamics of two renal myxozoans in hatchery reared fry and juvenile Atlantic cod / Gadus morhua L. Parasitology. 2010;137:1501鈥?3. CrossRef
  22. Jones S, Prosperi-Porta G, Dawe S, Taylor K, Goh B. / Parvicapsula minibicornis in anadromous sockeye ( / Oncorhynchus nerka) and coho ( / Oncorhynchus kisutch) salmon from tributaries of the Columbia River. J Parasitol. 2004;90:882鈥?. CrossRef
  23. Latorra D, Campbell K, Wolter A, Hurley JM. Enhanced allele-specific PCR discrimination in SNP genotyping using 3鈥?Locked Nucleic Acid (LNA) primers. Hum Mutat. 2003;22:79鈥?5. CrossRef
  
• 刊物主题：Parasitology; Infectious Diseases; Tropical Medicine; Entomology;
• 出版者：BioMed Central
• ISSN：1756-3305

文摘

Background Parvicapsula pseudobranchicola is a marine myxosporean parasite infecting farmed Atlantic salmon (Salmo salar). A major site for the parasite is the pseudobranch, which may be destroyed in heavily infected fish. Parvicapsulosis may be associated with significant mortality, although the main effect of infections seems to be runting. In situ hybridization (ISH) is, in the absence of specific antibodies, the preferred method for the detection of cell- and tissue tropisms of myxozoans in the early phases of infection of the host, and provides information about the possible association between the pathogen and pathology. A positive diagnosis of parvicapsulosis is based on histopathology and PCR. The aim of the present work was to develop a specific, sensitive and robust ISH assay for the detection of P. pseudobranchicola in tissues. Methods The ISH method was designed to specifically target P. pseudobranchicola 18S rDNA/rRNA using a locked nucleic acid (LNA) modified oligonucleotide probe. The method was tested on paraffin embedded P. pseudobranchicola infected pseudobranchs. The infections were confirmed by light microscopy revealing the presence of typical P. pseudobranchicola trophozoites and spores, and the presence of parasite was confirmed with real-time RT-PCR. Results Specific regions stained by ISH overlapped well with the parasitized and degenerated regions in neighbouring HE stained sections. No staining was observed in pseudobranchs of Atlantic salmon which had been held in P. pseudobranchicola-free water. Conclusions We report here the development of a sensitive ISH assay for the detection of P. pseudobranchicola in paraffin embedded tissue. The technique will be valuable in the study of host entry, early proliferation, pre-spore development, pathology and tissue tropism in Atlantic salmon.