Observation de marteilia spp.

Edition n° 1
Quantification of Perkinsus sp. infection intensity using
Ray’s Fluid Thioglycolate Medium (RFTM) Method
CONTENTS
Editions
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Ifremer, Genetic and Pathology Laboratory, Avenue de Mus de Loup, 17390 La Tremblade, France
Quantification of Perkinsus sp. infection intensity using
Ray’s Fluid Thioglycolate Medium (RFTM) Method
1. Scope
This procedure explains the technique used to quantify the protistan Perkinsus sp. in molluscs by using a special culture
medium. The present technique is adapted from Ray (1952).
2. References
• Council Directive 24 October 2006 on animal health requirements for aquaculture animals and
products thereof, and on the prevention and control of certain diseases in aquatic animals. • OIE. Manual of Diagnostic Tests for Aquatic Animals (last edition).
• Howard, D.W., E.J. Lewis, B.J. Keller, and C.S. Smith (2004). Histological techniques for marine bivalves
mollusks and crustaceans. NOAA Tech. Memo. NOS NCCOS 5, 218 p. • Villalba A., K.S. Reece, M.C. Ordás, S.M. Casas and A. Figueras (2004). Perkinsosis in molluscs: A review. Aquat.
• Ray, S.M. (1952). A culture technique for the diagnosis of infection with Dermocystidium marinum Mackin, Owell
and Collier in oysters. Science, 116, 360-361. • Bower, S.M. (2010): Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish: Perkinsus of
Clams and Cockles and Perkinsus marinus (“Dermo” disease) of oysters. 3. General information
Perkinsosis have been reported from many parts of the world (Europe, America, Asia, Australia) in different mollusc
species including abalone, oysters, clams, scallops, etc. (Garcia et al. in Villalba, 2008).
Perkinsus olseni is a pathogenic protistan infecting different species of clams in Europe (mainly Ruditapes decussatus and
R. philippinarum). It was also reported as an important pathogenic organism of the abalone Haliotis rubra in Australia.
Perkinsus marinus causes disease of economic importance in Crassostrea virginica. Crassostrea gigas can be infected to a
lesser extent. Perkinsus chesapeaki is a species commonly observed in USA in several clam species (e.g. Mya arenaria)
and the oyster C. virginica.
4. Equipment and medium preparation
4.1. Equipment
• Microscope with objective X10
4.2. Medium preparation
4.2.1. Thioglycolate medium • thioglycolate medium (Sigma T9032) : 29.4 g • sterile sea water 800 ml Adjust at pH 7, add enough sterile seawater for 1000 ml, and bring to the boil under stirring. Autoclave. 4.2.2. Antibiotic solution • penicillin G (Sigma P 3032): 6.66 g • sterile sea water 1000 ml Filter at 0.45µm and aliquote in 50 ml tubes. Keep frozen (- 20°C). Quantification of Perkinsus sp. by RFTM method
4.2.3. NaOH 2 M solution • NaOH : 80 g • sterile sea water 1000 ml Keep at room temperature. • dH2O : 1 litre Adjust pH to 7.2 and keep at 4°C. 5. Operating procedure
5.1. Procedure
1. Dispense 9 ml of thioglycolate medium per tube 2. Add 1 ml of antibiotic solution per tube 3. Open animals and discard internal liquid. Rinse knife and pliers with alcohol between each individual. 4. Collect the 4 gill leafs with a pinch, weight them, put them in a tube previously prepared and incubate them in the dark at room temperature for at least 1 week. 5. Centrifuge tubes at 1000 rpm for 10 minutes at room temperature
6. Discard 8 ml of supernatant
7. Add 8 ml of NaOH 2M solution and incubate at 50°C for 1 hour
8. Vortex, centrifuge tubes at 1000 rpm for 10 minutes at room temperature
9. Repeat the 2 precedent steps if tissue is not fully digested
10. Discard 8 ml of supernatant and add 8 ml of PBS 1X solution.
11. Vortex, centrifuge tubes at 1000 rpm for 10 minutes at room temperature
12. Discard most of the supernatant and keep only 2 ml of liquid
13. Mix vigorously samples before putting them on a haemocytometer
14. Count the cells under microscope (objective X10) four times on the entire haemocytometer or count the cells four
times on 10 rectangles (see picture page 4). If number of parasites is too high, add 1 or 2 ml of PBS 1X in the tube, mix and count again. 5.2. Count

Number of parasites / ml = 10 000 x ΣX / 4

Number of parasites / ml = 1000 x ΣX / 4

Number of parasites per gram of tissue = Number of parasites x V / Y
V: final volume of PBS in the tube (in ml) Y: gill weight in grams Quantification of Perkinsus sp. by RFTM method
Picture: Haemocytometer with a Perkinsus sp. culture in RFTM.
Quantification of Perkinsus sp. by RFTM method

Source: http://www.eurl-mollusc.eu/content/download/39253/536483/file/perkinsus_rftm_1.pdf