Diagnostic manual

Category C diseases

Marteilia refringens, Bonamia ostreae and Bonamia exitiosa

The following section provides recommendations on the methods to be used for the diagnosis of category C diseases (endemic) and links to corresponding SOPs.

1. General rules

Reference : Commission Delegated Regulation (EU) 2020/689 describes the rules for surveillance, eradication programmes, and disease-free status for certain listed and emerging diseases, Annexe VI provides specific requirement as regards diseases of aquatic animal.

• For surveillance programme / granting or maintaining free status
  • Histopathology, or tissue imprints or PCR
• To confirm of rule out suspicion
  • Histopathology, or tissue imprints or in situ Hybridization (ISH) AND PCR & sequencing
  • If no material for histopathology, or tissue imprints or ISH available: 2 PCR assays targeting different fragments of the genome & sequencing

2. Recommended methods

Reference: Commission Implementing Decision (EU) 2015/1554 of 11 September 2015 laying down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods and up-dates

Marteilia refringens	SOP
Histopathology	Marteilia sp. 2
Cytology
In Situ Hybridization M. refringens– Probe M2A / M3AS, Le Roux et al. 2001 (ITS1)	M.refringens_ISH_4
Conventional PCR M. refringens, Le Roux et al. 2001, Primers M2A & M3AS also named Pr4 & Pr5 (ITS1- 412bp). Type discrimination possible by RFLP.	Marteilia PCR-RFLP_3
Taqman real-time PCR M. refringens / Bonamia sp., Canier et al. 2020 (18S)	Real time PCR Marteilia refringens and Bonamia sp. 1
Taqman real-time PCR M. refringens type M / type O, EURL unpusblished (ITS1)	Real time PCR for the detection and typing of M. refringens 3
Recommended regions for sequencing: 18S and ITS1

Bonamia ostreae and Bonamia exitiosa	SOP
Histopathology	Bonamia sp. v5
Cytology
In Situ Hybridization Bonamia sp - Probe BO / BOAS, Cochennec et al. 2000 (18S)	Bonamia HIS v1
Conventionnal PCR Bonamia sp., Cochennec et al. 2000, primers BO/BOAS (18S - 304bp). Species discrimination possible by RFLP	Bonamia PCR-RFLP 2
SYBR Green PCR B. ostreae / B. exitiosa, Ramilo et al. 2013, primers BOSTRE-F/R BEXIT F/R (ITS -18S)	Not available
Taqman real-time PCR M. refringens / Bonamia sp., Canier et al. 2020 (18S)	Real time PCR Marteilia refringens and Bonamia sp. 1
Taqman real-time PCR B.ostreae/ B.exitiosa, EURL unpusblished (Actin)	B.ostreae and B.exitiosa RT PCR
Recommended regions for sequencing: 18S and Actin

Category A diseases

The following section provides recommendations on the methods to be used for the diagnosis of category A diseases (exotic) and links to corresponding SOPs. Those recommendations are based on the the OIE (World Organisation for Animal Health) Manual of diagnostic Test for Aquatic Animals and EURL expertise.

Perkinsus marinus

1. General rules

• for surveillance programme / granting or maintaining free status
  • Histopathology, or Ray’s Fluid Thioglycolate Medium (RFTM) culture or PCR
• To confirm of rule out suspicion
  • Histopathology, or Ray’s Fluid Thioglycolate Medium (RFTM) or in situ Hybridization (ISH) AND PCR & sequencing ITS

2. Recommended methods

Perkinsus marinus	SOP
Histopathology	Perkinsus sp. 1
Ray’s Fluid Thioglycolate Medium (RFTM) culture	Perkinsus RFTM 1
In Situ Hybrization P. marinus, Probe PmarLSU-181DIG, Moss et al. 2006 (LSU)	P.marinus_ISH_1
SYBR Green PCR P. Marinus, Audemard et al. 2004, Primers PmarITS-70F & PmarITS600R (ITS-509bp)	Pmarinus_RealTimePCR_1

Mikrocytos mackini

1. General rules

• For surveillance programme / granting or maintaining free status
  • Histopathology or PCR
• To confirm of rule out suspicion
  • Histopathology or  in situ Hybridization (ISH) AND PCR & sequencing on ITS + 18S

2. Recommended methods

Mikrocytos mackini	SOP
Histopathology	Mikrocytos sp. 2
In Situ Hybrization M. mackini, Probe MACKINI-1 F&R, Meyer et al. 2005 (18S)	M.mackini_ISH_1
Conventional PCR M. mackini, Carnegie et al. 2003, Primers MIKROCYTOS-F&R (18S-546 bp )	Mmackini_PCR_1
Taqman Real Time PCR M. mackini, Polinski et al., 2015 (ITS2)	Mmackini_RealTimePCR_1