Research Article | Volume: 5, Issue: 3, May-June, 2017

Utilization of molecular methods to relate Red Mark Syndrome affecting Oncorhynchus mykiss to an unculturable Rickettsiales organism

Francesca Cecchini Marisa Manzano Lucilla Iacumin Giuseppe Comi Donatella Volpatti Elena Galletti Marco Galeotti   

Open Access   

Published:  Jun 19, 2017

DOI: 10.7324/JABB.2017.50301

Red Mark Syndrome (RMS) is an inflammatory skin condition related to the presence of an unculturable Rickettsiales organism that affects farmed rainbow trout, Oncorhynchus mykiss. This disease is responsible of economic losses for fish farmers in Europe. The aim of this work was to confirm the presence of Rickettsiales organisms in affected fish and to develop a specific and sensitive system that can detect the unculturable Rickettsiales organisms in farmed trout. Trout gathered from affected and unaffected farms were initially submitted to necropsy and subsequently tissue samples were collected from each fish. Specific primers for a nested PCR (RiFCfw and RiFC), and a DNA probe for dot blot assay were designed using the 16S rRNA sequence. The use of the DNA probe to target amplicons as the template increased the sensitivity to 0.5 pg/µl DNA. Fifteen trout were analysed, of which nine showed visible skin lesions. Eight trout out of the nine with skin lesions were positive for the presence of the unculturable Rickettsiales organism DNA sequence. The tests developed can help rainbow trout producers to quickly treat fish in order to reduce economic losses. Up to 30% of fish with skin lesions are downgraded and rejected at the market by consumers.

Keyword:     RickettsialesRed Mark Syndromefarmed rainbow troutPCRdot blotDNA probes.


Cecchini F, Manzano M, Iacumin L, Comi G, Volpatti D, Galletti E, Galeotti M. Utilization of molecular methods to relate Red Mark Syndrome affecting Oncorhynchus mykiss to anunculturable Rickettsiales organism. J App Biol Biotech. 2017; 5 (03): 001-007. DOI: 10.7324/JABB.2017.50301

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text

1. Ferguson HW. Skin. In: Ferguson HW editors. Systemic Pathology of Fish. London, Scotian Press; 2006, p. 64-89.

2. Metselaar M, Thompson KD, Gratacap RML, Kik MJL, LaPatra SE, Lloyd SJ, Call RD, Smith PD, Adams A. Association of red-mark syndrome with a Rickettsia-like organism and its connection with strawberry disease in the USA. Journal of Fish Disase. 2010; 33:849-858.

3. Schmidt-Posthaus H, Bergmann W, Knüsel R, Heistinger H, Licek E. Appearance of red mark syndrome /cold water strawberry disease in Switzerland and Austria. Diseases of Aquatic Organisms. 2009; 88:65–68.

4. Verner-Jeffreys DW, Pond MJ, Peeler EJ, Rimmer GSE, Oidtmann B, Way K, Mewett J, Jeffrey K, Bateman K, Reese RA, Feist SW. Emergence of cold water strawberry disease of rainbow trout Oncorynchus mykiss in England and Wales: outbreak investigations and transmission studies. Diseases of Aquatic Organisms. 2008; 79:207-218.

5. Galeotti M, Giavenni R, Volpatti D, Beraldo P, Feist S. Red mark syndrome/cold water strawberry disease: emergence in Italy and histopathological investigations. 15th international conference on diseases of fish and shellfish organized by the EAFP. 2011. Split, Croatia.

6. Lloyd SJ, La Patra SE, Snekvik KR, Cain KD, Call DR. Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout. Oncorhynchus mykiss (Walbaum). Journal of Fish Disease. 2011; 34:701-709.

7. McCarthy Ú, Casadei E, Wang T, Secombes CJ. Red mark syndrome in rainbow trout Oncorhynchus mykiss: Investigation of immune responses in lesions using histology, immunohistochemistry and analysis of immune gene expression. Fish & Shellfish Immunology. 2013; 34:1119 - 1130.

8. Oidtmann B, LaPatra SE, Verner-Jeffreys D, Pond M, Peeler EJ, Noguera EP, Bruno DW, St-Hilaire S, Schubiger CB, Snekvik K, Crumlish M, Green DM, Metselaar M, Rodger H, Schmidt-Posthaus H, Galeotti M, Feist SW. Differential characterization of emerging skin diseases of rainbow trout – a standardized approach to capturing disease characteristics and development of case definitions. Journal of Fish Disease. 2013; 36:921-937.

9. Olson DP, Beleau MH, Busch RA, Roberts S, Krieger RI. Strawberry disease in rainbow trout, Salmo gairdneri Richardson. Journal Fish Disease 1985; 8:103–111.

10. Lloyd SJ, LaPatra SE, Snekvik KR, St-Hilaire S, Cain KD, Call DR. Strawberry disease lesions in rainbow trout from southern Idaho are associated with DNA from a Rickettsia-like organism. Diseases of Aquatic Organisms. 2008; 82:111-118.

11. Dumler JS, Barbet AF, Bekker, CP, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. International Journal Systematyc Evolutionary Microbiology. 2001; 51:2145-2165.

12. Yao DCJ, Moellering RCJr. Antibacterial agents. In: Murray PR editors, Manual of clinical microbiology, Washington DC: ASM Press; 2003, p. 1039-1073.

13. Langendijl PS, Schut F, Jansen GJ, Raangs GC, Kamphuis GR, Wilkinson MH, Welling GW. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in faecal samples. Applied Environmental Microbiology. 1995; 61:3069–3075.

14. Manzano M, Iacumin L, Giusto C, Cecchini F, Patthey C, Fontanillas R, Comi G. Utilization of denaturing gradient gel electrophoresis (DGGE) to evaluate the intestinal microbiota of brown trout Salmo trutta fario. Journal Veteterinary Science Medical Diagnostic. 2013;1: 1-6.

15. Kim DH, Brunt J, Austin B. Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss). Journal Applied Microbiology. 2007;102: 1654–1664.

16. Cecchini F, Iacumin L, Fontanot M, Comi G, Manzano M. Identification of the unculturable bacteria Candidatus Arthromitus in the intestinal content of trout using dot blot and Southern blot techniques. Veterinary Microbiology 2012; 156:384-394.

17. Manzano M, Giusto C, Iacumin L, Patthey C, Cecchini F, Fontanillas R, Comi G. Nested PCR for the detection of Candidatus arthromitus in fish. FEMS Microbiology Letters. 2010; 308:35–39.

18. Galeotti M, Volpatti D, Beraldo P, Brunetti B, Galletti E, Feist SW. Red mark syndrome in rainbow trout (Oncorhynchus mykiss) farmed in Italy: anatomo-histopathological investigations. Journal of Comparative Pathology. 2013; 148:54-54.

19. Manzano M, Cocolin L, Cantoni C, Comi G. Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides differentiation using a PCR-RE technique. International Journal of Food Microbiology. 2003; 81:249-254.

20. Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acid Research. 1988; 16:10881–10890.

21. Jullien N. http://crn2; AmplifX 1.6.2; CNRS, Aix-Marseille Université. 2013.

22. Chen S, Fairley CK, Tabrizi SN, Quinn MA, Garland SM. Southern blot and dot blot hybridisation compared to PCR for the detection of human papillomavirus DNA in biopsies of the uterine cervix from women with dysplasia. Clinical Diagnostic Virology. 1993; 1: 187-194.

23. U. S. National Library of Medicine (2014); Blast.h

Article Metrics
153 Views 39 Downloads 192 Total



Related Search

By author names

Similar Articles